Non-human animals having a humanized programmed cell death 1 gene

ABSTRACT

Non-human animals, and methods and compositions for making and using the same, are provided, wherein the non-human animals comprise a humanization of a Programmed cell death 1 (Pdcd1) gene. The non-human animals may be described, in some embodiments, as having a genetic modification to an endogenous Pdcd1 gene so that the non-human animals express a PD-1 polypeptide that includes a human portion and an endogenous portion (e.g., a non-human portion).

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority of U.S. ProvisionalApplication Nos. 62/014,181 filed Jun. 19, 2014, 62/086,518 filed Dec.2, 2014, and 62/138,221 filed Mar. 25, 2015, the entire contents ofwhich are incorporated herein by reference.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

The Sequence Listing in an ASCII text file, named 31969_SEQ.txt of 23KB, created on Jun. 4, 2015, and submitted to the United States Patentand Trademark Office via EFS-Web, is incorporated herein by reference.

BACKGROUND

Although an intense focus of medical research and development has beendevoted to cancer immunotherapy and significant improvements have beenmade, cancer remains a major challenge in the healthcare industryworldwide. This major challenge is due, in part, to the ability ofcancer cells to evade the monitoring mechanisms of the immune system,which is partly the result of inhibition and/or down-regulation ofanti-tumor immunity. Still, development of in vivo systems to optimallydetermine the therapeutic potential of new cancer therapies that aredesigned to activate and/or promote anti-tumor immunity and determinethe molecular aspects of how cancer cells provide inhibitory signals toimmune cells (e.g., T cells) is lacking. Such systems provide a sourcefor assays for assessing the therapeutic efficacy of candidate agentsthat promote an anti-tumor environment in vivo.

SUMMARY

The present invention encompasses the recognition that it is desirableto engineer non-human animals to permit improved systems for identifyingand developing new therapeutics that can be used for the treatment ofcancer. The present invention also encompasses the recognition that itis desirable to engineer non-human animals to permit improved systemsfor identifying and developing new therapeutics that can be used totreat autoimmune (or inflammatory) diseases, disorders or conditions.Further, the present invention also encompasses the recognition thatnon-human animals having a humanized Pdcd1 gene and/or otherwiseexpressing, containing, or producing a human or humanized PD-1polypeptide are desirable, for example for use in identifying anddeveloping cancer therapeutics that up-regulate anti-tumor immunity. Insome embodiments, non-human animals of the present invention provideimproved in vivo systems for the identification and development ofcombination therapies that include targeting PD-1.

In some embodiments, the present invention provides a non-human animalhaving a genome comprising a Pdcd1 gene that includes genetic materialfrom two different species (e.g., a human and a non-human). In someembodiments, the Pdcd1 gene of a non-human animal as described hereinencodes a PD-1 polypeptide that contains human and non-human portions,wherein the human and non-human portions are linked together and form afunctional PD-1 polypeptide. In some embodiments, the Pdcd1 gene of anon-human animal as described herein encodes a PD-1 polypeptide thatcontains an extracellular domain, in whole or in part, of a human PD-1polypeptide.

In some embodiments, the present invention provides a non-human animalthat expresses a PD-1 polypeptide, which PD-1 polypeptide comprises ahuman portion and an endogenous portion. In some embodiments, a PD-1polypeptide of the present invention is translated in a cell of thenon-human animal with a non-human signal peptide; in some certainembodiments, a rodent signal peptide.

In some embodiments, an endogenous portion comprises an intracellularportion of an endogenous PD-1 polypeptide. In some embodiments, anendogenous portion further comprises a transmembrane portion of anendogenous PD-1 polypeptide. In some embodiments, an endogenous portionhas an amino acid sequence that is at least 50%, at least 60%, at least70%, at least 80%, at least 90%, or at least 95% identical to acorresponding amino acid sequence of a mouse PD-1 polypeptide thatappears in FIG. 8. In some embodiments, an endogenous portion has anamino acid sequence that is substantially identical to a correspondingamino acid sequence of a mouse PD-1 polypeptide that appears in FIG. 8.In some embodiments, an endogenous portion has an amino acid sequencethat is identical to a corresponding amino acid sequence of a mouse PD-1polypeptide that appears in FIG. 8.

In some embodiments, a human portion comprises amino acids 35-145,27-145, 27-169, 26-169 or 21-170 of a human PD-1 polypeptide. In someembodiments, a human portion comprises an amino acid sequence that is atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, or atleast 95% identical to a corresponding amino acid sequence of a humanPD-1 polypeptide that appears in FIG. 8. In some embodiments, a humanportion comprises an amino acid sequence that is substantially identicalto a corresponding amino acid sequence of a human PD-1 polypeptide thatappears in FIG. 8. In some embodiments, a human portion comprises anamino acid sequence that is identical to a corresponding amino acidsequence of a human PD-1 polypeptide that appears in FIG. 8.

In some embodiments, a PD-1 polypeptide, which comprises a human portionand an endogenous portion, is encoded by an endogenous Pdcd1 gene. Insome certain embodiments, an endogenous Pdcd1 gene comprises endogenousPdcd1 exons 1, 4 and 5. In some certain embodiments, an endogenous Pdcd1gene further comprises an endogenous Pdcd1 exon 3 in whole or in part.In some certain embodiments, an endogenous Pdcd1 gene comprises SEQ IDNO:21. In some certain embodiments, an endogenous Pdcd1 gene comprisesSEQ ID NO:22. In some certain embodiments, an endogenous Pdcd1 genecomprises SEQ ID NO:21 and SEQ ID NO:22.

In some embodiments, a PD-1 polypeptide expressed by a non-human animalas described herein has an amino acid sequence that is at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, or at least 95%identical to SEQ ID NO:6. In some embodiments, a PD-1 polypeptideexpressed by a non-human animal as described herein has an amino acidsequence that is substantially identical to SEQ ID NO:6. In someembodiments, a PD-1 polypeptide expressed by a non-human animal asdescribed herein has an amino acid sequence that is identical to SEQ IDNO:6.

In some embodiments, the present invention provides a humanized Pdcd1locus comprising one or more exons of a non-human Pdcd1 gene operablylinked to one or more exons, in whole or in part, of a human Pdcd1 gene.In some embodiments, a humanized Pdcd1 locus further comprises 5′ and 3′non-human Pdcd1 untranslated regions (UTRs) flanking the one or moreexons of a human Pdcd1 gene. In some embodiments, a humanized Pdcd1locus is under the control of a rodent promoter; in some certainembodiments, an endogenous rodent promoter.

In some embodiments, a humanized Pdcd1 locus comprises non-human Pdcd1exons 1, 3, 4 and 5 operably linked to a human Pdcd1 exon 2. In someembodiments, a humanized Pdcd1 locus comprises non-human Pdcd1 exons 1,4 and 5, a human Pdcd1 exon 2 and further comprises a Pdcd1 exon 3,which Pdcd1 exon 3 comprises a human portion and a non-human portion,and wherein said non-human and human exons are operably linked. In someembodiments, a human portion of a Pdcd1 exon 3 includes nucleotides thatencode a PD-1 stalk sequence. In some embodiments, a human portion of aPdcd1 exon 3 includes about 71 bp of a human Pdcd1 exon 3. In someembodiments, a non-human portion of a Pdcd1 exon 3 includes nucleotidesthat encode a transmembrane sequence. In some embodiments, a non-humanportion of a Pdcd1 exon 3 includes about 91 bp of a rodent Pdcd1 exon 3.

In some embodiments, the present invention provides a non-human animalcomprising a Pdcd1 gene that comprises an endogenous portion and a humanportion, where the endogenous and human portions are operably linked toa rodent Pdcd1 promoter. In some embodiments, the rodent Pdcd1 promoteris an endogenous rodent Pdcd1 promoter.

In some embodiments, an endogenous portion comprises endogenous Pdcd1exons 1, 4 and 5. In some embodiments, an endogenous portion furthercomprises endogenous Pdcd1 exon 3 in whole or in part. In someembodiments, exons 1, 3 in whole or in part, 4 and 5 of an endogenousPdcd1 gene are at least 50%, at least 60%, at least 70%, at least 80%,at least 90%, or at least 95% identical to the corresponding exons 1, 3in whole or in part, 4 and 5 of an endogenous Pdcd1 gene that appears inFIG. 8. In some embodiments, exons 1, 3 in whole or in part, 4 and 5 ofan endogenous Pdcd1 gene are at substantially identical to thecorresponding exons 1, 3 in whole or in part, 4 and 5 of an endogenousPdcd1 gene that appears in FIG. 8. In some embodiments, exons 1, 3 inwhole or in part, 4 and 5 of an endogenous Pdcd1 gene are at identicalto the corresponding exons 1, 3 in whole or in part, 4 and 5 of anendogenous Pdcd1 gene that appears in FIG. 8.

In some embodiments, a human portion encodes amino acids 21-170, 26-169,27-169, 27-145 or 35-145 of a human PD-1 polypeptide.

In some embodiments, a human portion comprises exon 2 of a human Pdcd1gene. In some embodiments, a human portion further comprises a humanPdcd1 exon 3 in whole or in part. In some embodiments, human Pdcd1 exons2 and 3, in whole or in part, are at least 50%, at least 60%, at least70%, at least 80%, at least 90%, or at least 95% identical to thecorresponding exons 2 and 3, in whole or in part, of a human Pdcd1 genethat appears in FIG. 8. In some embodiments, human Pdcd1 exons 2 and 3,in whole or in part, are substantially identical to the correspondingexons 2 and 3, in whole or in part, of a human Pdcd1 gene that appearsin FIG. 8. In some embodiments, human Pdcd1 exons 2 and 3, in whole orin part, are identical to the corresponding exons 2 and 3, in whole orin part, of a human Pdcd1 gene that appears in FIG. 8. In someembodiments, a human portion comprises a sequence that iscodon-optimized for expression in a non-human animal; in someembodiments, expression in a rodent; in some certain embodiments,expression in a mouse; in some certain embodiments, expression in a rat.

In some embodiments, a human portion comprises a sequence that is atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, or atleast 95% identical to SEQ ID NO:23. In some embodiments, a humanportion comprises a sequence that is substantially identical to SEQ IDNO:23. In some embodiments, a human portion comprises a sequence that isidentical to SEQ ID NO:23. In some embodiments, a human portioncomprises SEQ ID NO:23.

In some embodiments, the present invention provides a PD-1 polypeptideproduced (or generated) by a non-human animal as described herein. Insome certain embodiments, a PD-1 polypeptide produced by a non-humananimal as described herein comprises an amino acid sequence that is atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, or atleast 95% identical to SEQ ID NO:6. In some certain embodiments, a PD-1polypeptide produced by a non-human animal as described herein comprisesan amino acid sequence that is substantially identical to SEQ ID NO:6.In some certain embodiments, a PD-1 polypeptide produced by a non-humananimal as described herein comprises an amino acid sequence that isidentical to SEQ ID NO:6.

In some embodiments, the present invention provides an isolated cell ortissue from a non-human animal as described herein. In some embodiments,the present invention provides an isolated cell or tissue that comprisesa Pdcd1 gene as described herein. In some embodiments, a cell is alymphocyte. In some embodiments, a cell is selected from a B cell,dendritic cell, macrophage, monocyte (e.g., an activated monocyte), NKcell, and T cell (e.g., an activated T cell). In some embodiments, atissue is selected from adipose, bladder, brain, breast, bone marrow,eye, heart, intestine, kidney, liver, lung, lymph node, muscle,pancreas, plasma, serum, skin, spleen, stomach, thymus, testis, ovum,and a combination thereof.

In some embodiments, the present invention provides a non-humanembryonic stem cell whose genome comprises a Pdcd1 gene as describedherein. In some embodiments, a non-human embryonic stem cell is a mouseembryonic stem cell and is from a 129 strain, C57BL/6 strain or a BALB/cstrain. In some embodiments, a non-human embryonic stem cell is a mouseembryonic stem cell and is from a 129 strain, C57BL/6 strain or amixture thereof. In some embodiments, a non-human embryonic stem cell isa mouse embryonic stem cell and is from a mixture of 129 and C57BL/6strains.

In some embodiments, a non-human embryonic stem cell has a genomecomprising a Pdcd1 gene that comprises SEQ ID NO:19, SEQ ID NO:20, SEQID NO:21, SEQ ID NO: 22 or a combination thereof.

In some embodiments, the present invention provides the use of anon-human embryonic stem cell as described herein to make a non-humananimal. In some certain embodiments, a non-human embryonic stem cell isa mouse embryonic stem cell and is used to make a mouse comprising aPdcd1 gene as described herein. In some certain embodiments, a non-humanembryonic stem cell is a rat embryonic stem cell and is used to make arat comprising a Pdcd1 gene as described herein.

In some embodiments, the present invention provides a non-human embryocomprising, made from, obtained from, or generated from a non-humanembryonic stem cell comprising a Pdcd1 gene as described herein. In somecertain embodiments, a non-human embryo is a rodent embryo; in someembodiments, a mouse embryo; in some embodiments, a rat embryo.

In some embodiments, the present invention provides the use of anon-human embryo as described herein to make a non-human animal. In somecertain embodiments, a non-human embryo is a mouse embryo and is used tomake a mouse comprising a Pdcd1 gene as described herein. In somecertain embodiments, a non-human embryo is a rat embryo and is used tomake a rat comprising a Pdcd1 gene as described herein.

In some embodiments, the present invention provides a targeting vector(or nucleic acid construct) as described herein. In some embodiments,the present invention provides a targeting vector (or nucleic acidconstruct) that comprises a humanized Pdcd1 gene as described herein. Insome embodiments, the present invention provides a targeting vector (ornucleic acid construct) that comprises a Pdcd1 gene that encodes a PD-1polypeptide that comprises a human extracellular domain in whole or inpart; in some certain embodiments a PD-1 polypeptide that comprisesamino acids 21-170, 26-169, 27-169, 27-145 or 35-145 of a human PD-1polypeptide.

In some embodiments, a targeting vector (or nucleic acid construct)comprises one or more exons, in whole or in part, of a non-human Pdcd1gene operably linked to one or more exons, in whole or in part, of ahuman Pdcd1 gene. In some embodiments, a targeting vector (or nucleicacid construct) comprises 5′ and 3′ non-human Pdcd1 untranslated regions(UTRs) flanking the one or more exons of a human Pdcd1 gene. In someembodiments, a targeting vector (or nucleic acid construct) comprisesone or more selection markers. In some embodiments, a targeting vector(or nucleic acid construct) comprises one or more site-specificrecombination sites. In some embodiments, a targeting vector (or nucleicacid construct) comprises a human Pdcd1 exon 2. In some embodiments, atargeting vector (or nucleic acid construct) comprises a human Pdcd1exon 2 and a human Pdcd1 exon 3 in whole or in part.

In some embodiments, the present invention provides use of a targetingvector (or nucleic acid construct) as described herein to make amodified non-human embryonic stem cell. In some embodiments, the presentinvention provides use of a targeting vector (or nucleic acid construct)as described herein to make a modified non-human embryo. In someembodiments, the present invention provides use of a targeting vector(or nucleic acid construct) as described herein to make a non-humananimal.

In some embodiments, the present invention provides a method of making anon-human animal that expresses a PD-1 polypeptide from an endogenousPdcd1 gene, wherein the PD-1 polypeptide comprises a human sequence, themethod comprising (a) inserting a genomic fragment into an endogenousPdcd1 gene in a rodent embryonic stem cell, said genomic fragmentcomprising a nucleotide sequence that encodes a human PD-1 polypeptidein whole or in part; (b) obtaining the rodent embryonic stem cellgenerated in (a); and, creating a rodent using the rodent embryonic stemcell of (b).

In some embodiments, a human sequence comprises amino acids 35-145,27-145, 27-169, 26-169 or 21-170 of a human PD-1 polypeptide.

In some embodiments, a nucleotide sequence comprises human Pdcd1 exon 2.In some embodiments, a nucleotide sequence further comprises human Pdcd1exon 3 in whole or in part. In some embodiments, a nucleotide sequencecomprises one or more selection markers. In some embodiments, anucleotide sequence comprises one or more site-specific recombinationsites.

In some embodiments, the present invention provides a method of making anon-human animal whose genome comprises a Pdcd1 gene that encodes a PD-1polypeptide having a human portion and an endogenous portion, whichportions are operably linked to a rodent Pdcd1 promoter, the methodcomprising modifying the genome of a non-human animal so that itcomprises a Pdcd1 gene that encodes a PD-1 polypeptide having a humanportion and an endogenous portion, which portions are operably linked toa rodent Pdcd1 promoter, thereby making said non-human animal.

In some embodiments, a rodent Pdcd1 promoter is an endogenous rodentPdcd1 promoter.

In some embodiments, a human portion comprises amino acids 35-145,27-145, 27-169, 26-169 or 21-170 of a human PD-1 polypeptide.

In some embodiments, a Pdcd1 gene is modified to include human Pdcd1exon 2. In some embodiments, a Pdcd1 gene is modified to include humanPdcd1 exon 2 and human Pdcd1 exon 3 in whole or in part.

In some embodiments, modifying the genome of a non-human animal isperformed in a non-human embryonic stem cell followed by generating anon-human animal with said non-human embryonic stem cell. In somecertain embodiments, the non-human embryonic stem cell is a rodentembryonic stem cell; in some embodiments, a mouse embryonic stem cell;in some embodiments, a rat embryonic stem cell.

In some embodiments, the present invention provides a non-human animalobtainable by methods as described herein.

In some embodiments, the present invention provides a method of reducingtumor growth in a non-human animal, the method comprising the steps ofadministering a drug targeting human PD-1 to a non-human animal whosegenome comprises a Pdcd1 gene that encodes a PD-1 polypeptide having ahuman portion and an endogenous portion, which portions are operablylinked to a rodent Pdcd1 promoter; the administering being performedunder conditions and for a time sufficient that tumor growth is reducedin the non-human animal.

In some embodiments, the present invention provides a method of killingtumor cells in a non-human animal, the method comprising the steps ofadministering a drug targeting human PD-1 to a non-human animal whosegenome comprises a Pdcd1 gene that encodes a PD-1 polypeptide having ahuman portion and an endogenous portion, which portions are operablylinked to a rodent Pdcd1 promoter; the administering being performedunder conditions and for a time sufficient that the drug mediateskilling of the tumor cells in the non-human animal.

In some embodiments, the present invention provides a method ofassessing the pharmacokinetic properties of a drug targeting human PD-1,the method comprising the steps of administering the drug to a non-humananimal whose genome comprises a Pdcd1 gene that encodes a PD-1polypeptide having a human portion and an endogenous portion, whichportions are operably linked a rodent Pdcd1 promoter; and performing anassay to determine one or more pharmacokinetic properties of the drugtargeting human PD-1.

In many embodiments, a non-human animal as described herein is a rodentwhose genome includes a Pdcd1 gene that encodes a PD-1 polypeptidehaving a human portion and an endogenous portion, which portions areoperably linked to a rodent Pdcd1 promoter. In many embodiments, arodent Pdcd1 promoter is an endogenous rodent Pdcd1 promoter. In manyembodiments, a human portion comprises amino acids 35-145, 27-145,27-169, 26-169 or 21-170 of a human PD-1 polypeptide.

In some embodiments, a drug targeting human PD-1 is a PD-1 antagonist.In some embodiments, a drug targeting human PD-1 is a PD-1 agonist. Insome embodiments, a drug targeting human PD-1 is an anti-PD-1 antibody.In some embodiments, a drug targeting human PD-1 is administeredintravenously, intraperitoneally, or subcutaneously.

In some embodiments, the present invention provides a non-human animaltumor model, which non-human animal expresses a PD-1 polypeptidecomprising a human portion and an endogenous portion.

In some embodiments, the present invention provides a non-human animaltumor model, which non-human animal has a genome comprising a Pdcd1 genethat comprises an endogenous portion and a human portion, wherein theendogenous and human portions are operably linked to a non-human animalPdcd1 promoter.

In some embodiments, the present invention provides a non-human animaltumor model obtained by (a) providing a non-human animal whose genomecomprises a Pdcd1 gene that includes an endogenous portion and a humanportion, which endogenous and human portions are operatively linked to anon-human animal Pdcd1 promoter; and (b) implanting one or more tumorcells in the rodent of (a); thereby providing said non-human animaltumor model.

In some embodiments, a non-human animal tumor model of the presentinvention is a rodent tumor model. In some embodiments, a non-humananimal Pdcd1 promoter is a rodent Pdcd1 promoter.

In some embodiments, the present invention provides a method foridentification or validation of a drug or vaccine, the method comprisingthe steps of delivering a drug or vaccine to a non-human animal whosegenome includes a Pdcd1 gene that encodes a PD-1 polypeptide, which PD-1polypeptide comprises a human portion and an endogenous portion, andmonitoring one or more of the immune response to the drug or vaccine,the safety profile of the drug or vaccine, or the effect on a disease,disorder or condition. In some embodiments, monitoring the safetyprofile includes determining if the non-human animal exhibits a sideeffect or adverse reaction as a result of delivering the drug orvaccine. In some embodiments, a side effect or adverse reaction isselected from morbidity, mortality, alteration in body weight,alteration of the level of one or more enzymes (e.g., liver), alterationin the weight of one or more organs, loss of function (e.g., sensory,motor, organ, etc.), increased susceptibility to one or more diseases,alterations to the genome of the non-human animal, increase or decreasein food consumption and complications of one or more diseases. In someembodiments, the disease, disorder or condition is induced in thenon-human animal. In some embodiments, the disease, disorder orcondition induced in the non-human animal is associated with a disease,disorder or condition suffered by one or more human patients in need oftreatment. In some certain embodiments, the drug is an antibody.

In some embodiments, the present invention provides use of a non-humananimal as described herein in the development of a drug or vaccine foruse in medicine, such as use as a medicament.

In some embodiments, the present invention provides use of a non-humananimal as described herein in the manufacture of a medicament for thetreatment of cancer, neoplasm, an infectious disease, an inflammatorydisease, disorder or condition, or an autoimmune disease, disorder orcondition.

In various embodiments, a Pdcd1 gene of the present invention includes aPdcd1 gene as described herein. In various embodiments, a Pdcd1 gene ofthe present invention encodes a PD-1 polypeptide having a human portionand an endogenous portion, which portions are operably linked to arodent Pdcd1 promoter. In various embodiments, a rodent promoter is anendogenous rodent promoter. In various embodiments, a human portioncomprises a human Pdcd1 exon 2. In various embodiments, a human portioncomprises a human Pdcd1 exon 2 and further comprises a human Pdcd1 exon3 in whole or in part.

In various embodiments, a PD-1 polypeptide of the present inventionincludes a PD-1 polypeptide as described herein. In various embodiments,a non-human animal of the present invention does not detectably expressa full-length endogenous non-human PD-1 polypeptide. In variousembodiments, a non-human animal of the present invention does notdetectably express an extracellular portion of an endogenous PD-1polypeptide. In various embodiments, a non-human animal of the presentinvention does not detectably express an N-terminal immunoglobulin Vdomain of an endogenous PD-1 polypeptide.

In various embodiments, a non-human animal of the present invention is arodent; in some embodiments, a mouse; in some embodiments, a rat. Insome embodiments, a mouse of the present invention is selected from thegroup consisting of a 129 strain, a BALB/C strain, a C57BL/6 strain, anda mixed 129×C57BL/6 strain; in some certain embodiments, 50% 129 and 50%C57BL/6; in some certain embodiments, 25% 129 and 75% C57BL/6.

As used in this application, the terms “about” and “approximately” areused as equivalents. Any numerals used in this application with orwithout about/approximately are meant to cover any normal fluctuationsappreciated by one of ordinary skill in the relevant art.

Other features, objects, and advantages of the present invention areapparent in the detailed description of certain embodiments thatfollows. It should be understood, however, that the detaileddescription, while indicating certain embodiments of the presentinvention, is given by way of illustration only, not limitation. Variouschanges and modifications within the scope of the invention will becomeapparent to those skilled in the art from the detailed description.

BRIEF DESCRIPTION OF THE DRAWING

The Drawing included herein, which is composed of the following Figures,is for illustration purposes only and not for limitation.

FIG. 1 shows a diagram, not to scale, of the genomic organization of anon-human (e.g., mouse) and human Programmed cell death 1 (Pdcd1) genes.Exons and untranslated regions (UTRs) are numbered beneath each exon andabove each UTR.

FIG. 2 shows a diagram, not to scale, of an exemplary method forhumanization of a non-human Programmed cell death 1 (Pdcd1) gene.Selected nucleotide junction locations are marked with a line below eachjunction. Sequences of these selected nucleotide junctions are indicatedby SEQ ID NOs.

FIG. 3 shows a diagram, not to scale, of the genomic organization of amouse and human Programmed cell death 1 (Pdcd1) genes indicating theapproximate locations of probes used in an assay described in Example 1.

FIG. 4 shows exemplary histograms of T cells gated on CD19 and CD8isolated from a wild-type mouse and a mouse heterozygous forhumanization of an endogenous Pdcd1 gene as described in Example 1 thatexpress mouse and/or humanized PD-1. Stimulated and unstimulated cellpopulations are indicated, as are cells stained with an isotype control.

FIG. 5 shows exemplary tumor growth curves over 21 days in micehomozygous for humanization of an endogenous Pdcd1 gene as described inExample 1. Control: antibody not specific for PD-1, a-hPD-1 Ab: antibodyspecific for human PD-1. Arrows indicate the days for antibodytreatment. The number of tumor-free mice on day 21 is shown for eachtreatment group.

FIG. 6 shows exemplary real-time PCR analysis of CD8b, CD3, IFN-g andPD-1 mRNA expression in spleens in mice homozygous for humanization ofan endogenous Pdcd1 gene as described in Example 1 after treatment withanti-PD-1 antibody. A, mean of five mice per group. B, expression levelsfor individual mice in each treatment group. Control: antibody notspecific for PD-1; α-PD-1: anti-PD-1 antibody.

FIG. 7 shows exemplary tumor growth curves over 60 days in micehomozygous for humanization of an endogenous Pdcd1 gene as described inExample 1 that were administered 0.3-25 mg/kg of anti-hPD-1 antibody or25 mg/kg of control antibody (antibody not specific for PD-1). Arrowsindicate the days of antibody treatment. The number of tumor-free miceon day 60 is shown for each treatment group.

FIG. 8 sets forth exemplary murine, human and humanized Pdcd1 and PD-1sequences, and an exemplary human nucleic acid sequence for humanizationof a non-human Pdcd1 gene. For mRNA sequences, bold font indicatescoding sequence and consecutive exons, where indicated, are separated byalternating underlined text; for humanized mRNA sequences, humansequences are contained within parentheses. For protein sequences,signal peptides are underlined, extracellular sequences are bold font,immunoglobulin V domain sequences are within parentheses, andintracellular sequences are italicized; and for humanized proteinsequences, non-human sequences are indicated in regular font, and humansequences are indicated in bold font.

DEFINITIONS

This invention is not limited to particular methods and experimentalconditions described herein, as such methods and conditions may vary. Itis also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention is defined bythe claims.

Unless defined otherwise, all terms and phrases used herein include themeanings that the terms and phrases have attained in the art, unless thecontrary is clearly indicated or clearly apparent from the context inwhich the term or phrase is used. Although any methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, particular methods andmaterials are now described. All publications mentioned herein arehereby incorporated by reference.

The term “approximately”, as applied herein to one or more values ofinterest, refers to a value that is similar to a stated reference value.In certain embodiments, the term “approximately” or “about” refers to arange of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%,13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less ineither direction (greater than or less than) of the stated referencevalue unless otherwise stated or otherwise evident from the context(except where such number would exceed 100% of a possible value).

The term “biologically active” includes a characteristic of any agentthat has activity in a biological system, in vitro or in vivo (e.g., inan organism). For instance, an agent that, when present in an organism,has a biological effect within that organism, is considered to bebiologically active. In particular embodiments, where a protein orpolypeptide is biologically active, a portion of that protein orpolypeptide that shares at least one biological activity of the proteinor polypeptide is typically referred to as a “biologically active”portion.

The term “comparable” includes to two or more agents, entities,situations, sets of conditions, etc. that may not be identical to oneanother but that are sufficiently similar to permit comparison betweenthem so that conclusions may reasonably be drawn based on differences orsimilarities observed. Those of ordinary skill in the art willunderstand, in context, what degree of identity is required in any givencircumstance for two or more such agents, entities, situations, sets ofconditions, etc. to be considered comparable.

The term “conservative”, e.g., as in a conservative amino acidsubstitution, includes substitution of an amino acid residue by anotheramino acid residue having a side chain R group with similar chemicalproperties (e.g., charge or hydrophobicity). In general, a conservativeamino acid substitution will not substantially change the functionalproperties of interest of a protein, for example, the ability of areceptor to bind to a ligand. Examples of groups of amino acids thathave side chains with similar chemical properties include: aliphaticside chains such as glycine, alanine, valine, leucine, and isoleucine;aliphatic-hydroxyl side chains such as serine and threonine;amide-containing side chains such as asparagine and glutamine; aromaticside chains such as phenylalanine, tyrosine, and tryptophan; basic sidechains such as lysine, arginine, and histidine; acidic side chains suchas aspartic acid and glutamic acid; and, sulfur-containing side chainssuch as cysteine and methionine. Conservative amino acids substitutiongroups include, for example, valine/leucine/isoleucine,phenylalanine/tyrosine, lysine/arginine, alanine/valine,glutamate/aspartate, and asparagine/glutamine. In some embodiments, aconservative amino acid substitution can be a substitution of any nativeresidue in a protein with alanine, as used in, for example, alaninescanning mutagenesis. In some embodiments, a conservative substitutionis made that has a positive value in the PAM250 log-likelihood matrixdisclosed in Gonnet et al. (1992) Exhaustive Matching of the EntireProtein Sequence Database, Science 256:1443-45, hereby incorporated byreference. In some embodiments, the substitution is a moderatelyconservative substitution wherein the substitution has a nonnegativevalue in the PAM250 log-likelihood matrix.

The term “control” includes the art-understood meaning of a “control”being a standard against which results are compared. Typically, controlsare used to augment integrity in experiments by isolating variables inorder to make a conclusion about such variables. In some embodiments, acontrol is a reaction or assay that is performed simultaneously with atest reaction or assay to provide a comparator. As used herein, a“control” may include a “control animal”. A “control animal” may have amodification as described herein, a modification that is different asdescribed herein, or no modification (i.e., a wild-type animal). In oneexperiment, the “test” (i.e., the variable being tested) is applied. Inthe second experiment, the “control,” the variable being tested is notapplied. In some embodiments, a control is a historical control (i.e.,of a test or assay performed previously, or an amount or result that ispreviously known). In some embodiments, a control is or comprises aprinted or otherwise saved record. A control may be a positive controlor a negative control.

The term “disruption” includes the result of a homologous recombinationevent with a DNA molecule (e.g., with an endogenous homologous sequencesuch as a gene or gene locus). In some embodiments, a disruption mayachieve or represent an insertion, deletion, substitution, replacement,missense mutation, or a frame-shift of a DNA sequence(s), or anycombination thereof. Insertions may include the insertion of entiregenes or fragments of genes, e.g., exons, which may be of an originother than the endogenous sequence (e.g., a heterologous sequence). Insome embodiments, a disruption may increase expression and/or activityof a gene or gene product (e.g., of a protein encoded by a gene). Insome embodiments, a disruption may decrease expression and/or activityof a gene or gene product. In some embodiments, a disruption may altersequence of a gene or an encoded gene product (e.g., an encodedprotein). In some embodiments, a disruption may truncate or fragment agene or an encoded gene product (e.g., an encoded protein). In someembodiments, a disruption may extend a gene or an encoded gene product;in some such embodiments, a disruption may achieve assembly of a fusionprotein. In some embodiments, a disruption may affect level but notactivity of a gene or gene product. In some embodiments, a disruptionmay affect activity but not level of a gene or gene product. In someembodiments, a disruption may have no significant effect on level of agene or gene product. In some embodiments, a disruption may have nosignificant effect on activity of a gene or gene product. In someembodiments, a disruption may have no significant effect on either levelor activity of a gene or gene product.

The terms “determining”, “measuring”, “evaluating”, “assessing”,“assaying” and “analyzing” are used interchangeably to refer to any formof measurement, and include determining if an element is present or not.These terms include both quantitative and/or qualitative determinations.Assaying may be relative or absolute. “Assaying for the presence of” canbe determining the amount of something present and/or determiningwhether or not it is present or absent.

The term “dosing regimen” or “therapeutic regimen” includes a set ofunit doses, in some embodiments, more than one, that are administeredindividually to a subject, typically separated by periods of time. Insome embodiments, a given therapeutic agent has a recommended dosingregiment, which may involve one or more doses. In some embodiments, adosing regimen comprises a plurality of doses each of which areseparated from one another by a time period of the same length; in someembodiments, a dosing regimen comprises a plurality of doses and atleast two different time periods separating individual doses.

The phrase “endogenous locus” or “endogenous gene” includes a geneticlocus found in a parent or reference organism. In some embodiments, theendogenous locus has a sequence found in nature. In some embodiments,the endogenous locus is a wild type locus. In some embodiments, thereference organism is a wild-type organism. In some embodiments, thereference organism is an engineered organism. In some embodiments, thereference organism is a laboratory-bred organism (whether wild-type orengineered).

The phrase “endogenous promoter” includes a promoter that is naturallyassociated, e.g., in a wild-type organism, with an endogenous gene.

The term “heterologous” includes an agent or entity from a differentsource. For example, when used in reference to a polypeptide, gene, orgene product or present in a particular cell or organism, the termclarifies that the relevant polypeptide, gene, or gene product: 1) wasengineered by the hand of man; 2) was introduced into the cell ororganism (or a precursor thereof) through the hand of man (e.g., viagenetic engineering); and/or 3) is not naturally produced by or presentin the relevant cell or organism (e.g., the relevant cell type ororganism type).

The term “host cell” includes a cell into which a heterologous (e.g.,exogenous) nucleic acid or protein has been introduced. Persons of skillupon reading this disclosure will understand that such terms refer notonly to the particular subject cell, but also is used to refer to theprogeny of such a cell. Because certain modifications may occur insucceeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term “host cell”.In some embodiments, a host cell is or comprises a prokaryotic oreukaryotic cell. In general, a host cell is any cell that is suitablefor receiving and/or producing a heterologous nucleic acid or protein,regardless of the Kingdom of life to which the cell is designated.Exemplary cells include those of prokaryotes and eukaryotes (single-cellor multiple-cell), bacterial cells (e.g., strains of E. coli, Bacillusspp., Streptomyces spp., etc.), mycobacteria cells, fungal cells, yeastcells (e.g., S. cerevisiae, S. pombe, P. pastoris, P. methanolica,etc.), plant cells, insect cells (e.g., SF-9, SF-21,baculovirus-infected insect cells, Trichoplusia ni, etc.), non-humananimal cells, human cells, or cell fusions such as, for example,hybridomas or quadromas. In some embodiments, the cell is a human,monkey, ape, hamster, rat, or mouse cell. In some embodiments, the cellis eukaryotic and is selected from the following cells: CHO (e.g., CHOK1, DXB-11 CHO, Veggie-CHO), COS (e.g., COS-7), retinal cell, Vero, CV1,kidney (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK), HeLa, HepG2,WI38, MRC 5, Colo205, HB 8065, HL-60, (e.g., BHK21), Jurkat, Daudi, A431(epidermal), CV-1, U937, 3T3, L cell, C127 cell, SP2/0, NS-0, MMT060562, Sertoli cell, BRL 3A cell, HT1080 cell, myeloma cell, tumorcell, and a cell line derived from an aforementioned cell. In someembodiments, the cell comprises one or more viral genes, e.g., a retinalcell that expresses a viral gene (e.g., a PER.C6™ cell). In someembodiments, a host cell is or comprises an isolated cell. In someembodiments, a host cell is part of a tissue. In some embodiments, ahost cell is part of an organism.

The term “humanized” includes nucleic acids or proteins whose structures(i.e., nucleotide or amino acid sequences) include portions thatcorrespond substantially or identically with structures of a particulargene or protein found in nature in a non-human animal, and also includeportions that differ from that found in the relevant particularnon-human gene or protein and instead correspond more closely withcomparable structures found in a corresponding human gene or protein. Insome embodiments, a “humanized” gene is one that encodes a polypeptidehaving substantially the amino acid sequence as that of a humanpolypeptide (e.g., a human protein or portion thereof—e.g.,characteristic portion thereof). To give but one example, in the case ofa membrane receptor, a “humanized” gene may encode a polypeptide havingan extracellular portion, in whole or in part, having an amino acidsequence as that of a human extracellular portion and the remainingsequence as that of a non-human (e.g., mouse) polypeptide. In someembodiments, a humanized gene comprises at least a portion of a DNAsequence of a human gene. In some embodiment, a humanized gene comprisesan entire DNA sequence of a human gene. In some embodiments, a humanizedprotein comprises a sequence having a portion that appears in a humanprotein. In some embodiments, a humanized protein comprises an entiresequence of a human protein and is expressed from an endogenous locus ofa non-human animal that corresponds to the homolog or ortholog of thehuman gene.

The term “identity”, e.g., as in connection with a comparison ofsequences, includes identity as determined by a number of differentalgorithms known in the art that can be used to measure nucleotideand/or amino acid sequence identity. In some embodiments, identities asdescribed herein are determined using a ClustalW v. 1.83 (slow)alignment employing an open gap penalty of 10.0, an extend gap penaltyof 0.1, and using a Gonnet similarity matrix (MACVECTOR™ 10.0.2,MacVector Inc., 2008).

The term “isolated” includes a substance and/or entity that has been (1)separated from at least some of the components with which it wasassociated when initially produced (whether in nature and/or in anexperimental setting), and/or (2) designed, produced, prepared, and/ormanufactured by the hand of man. Isolated substances and/or entities maybe separated from about 10%, about 20%, about 30%, about 40%, about 50%,about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%,or more than about 99% of the other components with which they wereinitially associated. In some embodiments, isolated agents are about80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%,about 95%, about 96%, about 97%, about 98%, about 99%, or more thanabout 99% pure. A substance is “pure” if it is substantially free ofother components. In some embodiments, as will be understood by thoseskilled in the art, a substance may still be considered “isolated” oreven “pure”, after having been combined with certain other componentssuch as, for example, one or more carriers or excipients (e.g., buffer,solvent, water, etc.); in such embodiments, percent isolation or purityof the substance is calculated without including such carriers orexcipients. To give but one example, in some embodiments, a biologicalpolymer such as a polypeptide or polynucleotide that occurs in nature isconsidered to be “isolated” when: a) by virtue of its origin or sourceof derivation is not associated with some or all of the components thataccompany it in its native state in nature; b) it is substantially freeof other polypeptides or nucleic acids of the same species from thespecies that produces it in nature; or c) is expressed by or isotherwise in association with components from a cell or other expressionsystem that is not of the species that produces it in nature. Thus, forinstance, in some embodiments, a polypeptide that is chemicallysynthesized or is synthesized in a cellular system different from thatwhich produces it in nature is considered to be an “isolated”polypeptide. Alternatively or additionally, in some embodiments, apolypeptide that has been subjected to one or more purificationtechniques may be considered to be an “isolated” polypeptide to theextent that it has been separated from other components: a) with whichit is associated in nature; and/or b) with which it was associated wheninitially produced.

The phrase “non-human animal” includes any vertebrate organism that isnot a human. In some embodiments, a non-human animal is a cyclostome, abony fish, a cartilaginous fish (e.g., a shark or a ray), an amphibian,a reptile, a mammal, and a bird. In some embodiments, a non-human mammalis a primate, a goat, a sheep, a pig, a dog, a cow, or a rodent. In someembodiments, a non-human animal is a rodent such as a rat or a mouse.

The phrase “nucleic acid” includes any compound and/or substance that isor can be incorporated into an oligonucleotide chain. In someembodiments, a “nucleic acid” is a compound and/or substance that is orcan be incorporated into an oligonucleotide chain via a phosphodiesterlinkage. As will be clear from context, in some embodiments, “nucleicacid” includes individual nucleic acid residues (e.g., nucleotidesand/or nucleosides); in some embodiments, “nucleic acid” includes anoligonucleotide chain comprising individual nucleic acid residues. Insome embodiments, a “nucleic acid” is or comprises RNA; in someembodiments, a “nucleic acid” is or comprises DNA. In some embodiments,a “nucleic acid” is, comprises, or consists of one or more naturalnucleic acid residues. In some embodiments, a “nucleic acid” is,comprises, or consists of one or more nucleic acid analogs. In someembodiments, a nucleic acid analog differs from a “nucleic acid” in thatit does not utilize a phosphodiester backbone. For example, in someembodiments, a “nucleic acid” is, comprises, or consists of one or more“peptide nucleic acids”, which are known in the art and have peptidebonds instead of phosphodiester bonds in the backbone, are consideredwithin the scope of the present invention. Alternatively oradditionally, in some embodiments, a “nucleic acid” has one or morephosphorothioate and/or 5′-N-phosphoramidite linkages rather thanphosphodiester bonds. In some embodiments, a “nucleic acid” is,comprises, or consists of one or more natural nucleosides (e.g.,adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine,deoxythymidine, deoxyguanosine, and deoxycytidine). In some embodiments,a “nucleic acid” is, comprises, or consists of one or more nucleosideanalogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine,pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C-5propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine,C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine,C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine,7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine,O(6)-methylguanine, 2-thiocytidine, methylated bases, intercalatedbases, and combinations thereof). In some embodiments, a “nucleic acid”comprises one or more modified sugars (e.g., 2′-fluororibose, ribose,2′-deoxyribose, arabinose, and hexose) as compared with those in naturalnucleic acids. In some embodiments, a “nucleic acid” has a nucleotidesequence that encodes a functional gene product such as an RNA orprotein. In some embodiments, a “nucleic acid” includes one or moreintrons. In some embodiments, a “nucleic acid” is prepared by one ormore of isolation from a natural source, enzymatic synthesis bypolymerization based on a complementary template (in vivo or in vitro),reproduction in a recombinant cell or system, and chemical synthesis. Insome embodiments, a “nucleic acid” is at least 3, 4, 5, 6, 7, 8, 9, 10,15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300,325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500,2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues long. In someembodiments, a “nucleic acid” is single stranded; in some embodiments, a“nucleic acid” is double stranded. In some embodiments, a “nucleic acid”has a nucleotide sequence comprising at least one element that encodes,or is the complement of a sequence that encodes, a polypeptide. In someembodiments, a “nucleic acid” has enzymatic activity.

The phrase “operably linked” includes a juxtaposition wherein thecomponents described are in a relationship permitting them to functionin their intended manner A control sequence “operably linked” to acoding sequence is ligated in such a way that expression of the codingsequence is achieved under conditions compatible with the controlsequences. “Operably linked” sequences include both expression controlsequences that are contiguous with the gene of interest and expressioncontrol sequences that act in trans or at a distance to control the geneof interest. The term “expression control sequence” includespolynucleotide sequences, which are necessary to effect the expressionand processing of coding sequences to which they are ligated.“Expression control sequences” include: appropriate transcriptioninitiation, termination, promoter and enhancer sequences; efficient RNAprocessing signals such as splicing and polyadenylation signals;sequences that stabilize cytoplasmic mRNA; sequences that enhancetranslation efficiency (i.e., Kozak consensus sequence); sequences thatenhance protein stability; and when desired, sequences that enhanceprotein secretion. The nature of such control sequences differsdepending upon the host organism. For example, in prokaryotes, suchcontrol sequences generally include promoter, ribosomal binding site,and transcription termination sequence, while in eukaryotes, typically,such control sequences include promoters and transcription terminationsequence. The term “control sequences” is intended to include componentswhose presence is essential for expression and processing, and can alsoinclude additional components whose presence is advantageous, forexample, leader sequences and fusion partner sequences.

The term “patient” or “subject” includes any organism to which aprovided composition is or may be administered, e.g., for experimental,diagnostic, prophylactic, cosmetic, and/or therapeutic purposes. Typicalpatients include animals (e.g., mammals such as mice, rats, rabbits,non-human primates, and/or humans). In some embodiments, a patient is anon-human animal. In some embodiments, a patient (e.g., a non-humananimal patient) may have a modification as described herein, amodification that is different as described herein or no modification(i.e., a wild-type non-human animal patient). In some embodiments, anon-human animal is suffering from or is susceptible to one or moredisorders or conditions. In some embodiments, a non-human animaldisplays one or more symptoms of a disorder or condition. In someembodiments, a non-human animal has been diagnosed with one or moredisorders or conditions.

The term “polypeptide” includes any polymeric chain of amino acids. Insome embodiments, a polypeptide has an amino acid sequence that occursin nature. In some embodiments, a polypeptide has an amino acid sequencethat does not occur in nature. In some embodiments, a polypeptide has anamino acid sequence that contains portions that occur in natureseparately from one another (i.e., from two or more different organisms,for example, human and non-human portions). In some embodiments, apolypeptide has an amino acid sequence that is engineered in that it isdesigned and/or produced through action of the hand of man.

The term “recombinant”, is intended to include polypeptides (e.g., PD-1polypeptides as described herein) that are designed, engineered,prepared, expressed, created or isolated by recombinant means, such aspolypeptides expressed using a recombinant expression vector transfectedinto a host cell, polypeptides isolated from a recombinant,combinatorial human polypeptide library (Hoogenboom H. R., (1997) TIBTech. 15:62-70; Azzazy H., and Highsmith W. E., (2002) Clin. Biochem.35:425-445; Gavilondo J. V., and Larrick J. W. (2002) BioTechniques29:128-145; Hoogenboom H., and Chames P. (2000) Immunology Today21:371-378), antibodies isolated from an animal (e.g., a mouse) that istransgenic for human immunoglobulin genes (see e.g., Taylor, L. D., etal. (1992) Nucl. Acids Res. 20:6287-6295; Kellermann S-A., and Green L.L. (2002) Current Opinion in Biotechnology 13:593-597; Little M. et al.(2000) Immunology Today 21:364-370; Murphy, A. J., et al. (2014) Proc.Natl. Acad. Sci. U.S.A 111(14):5153-5158) or polypeptides prepared,expressed, created or isolated by any other means that involves splicingselected sequence elements to one another. In some embodiments, one ormore of such selected sequence elements is found in nature. In someembodiments, one or more of such selected sequence elements is designedin silico. In some embodiments, one or more such selected sequenceelements result from mutagenesis (e.g., in vivo or in vitro) of a knownsequence element, e.g., from a natural or synthetic source. For example,in some embodiments, a recombinant polypeptide is comprised of sequencesfound in the genome of a source organism of interest (e.g., human,mouse, etc.). In some embodiments, a recombinant polypeptide has anamino acid sequence that resulted from mutagenesis (e.g., in vitro or invivo, for example in a non-human animal), so that the amino acidsequences of the recombinant polypeptides are sequences that, whileoriginating from and related to polypeptides sequences, may notnaturally exist within the genome of a non-human animal in vivo.

The term “replacement” includes a process through which a “replaced”nucleic acid sequence (e.g., a gene) found in a host locus (e.g., in agenome) is removed from that locus, and a different, “replacement”nucleic acid is located in its place. In some embodiments, the replacednucleic acid sequence and the replacement nucleic acid sequences arecomparable to one another in that, for example, they are homologous toone another and/or contain corresponding elements (e.g., protein-codingelements, regulatory elements, etc.). In some embodiments, a replacednucleic acid sequence includes one or more of a promoter, an enhancer, asplice donor site, a splice receiver site, an intron, an exon, anuntranslated region (UTR); in some embodiments, a replacement nucleicacid sequence includes one or more coding sequences. In someembodiments, a replacement nucleic acid sequence is a homolog of thereplaced nucleic acid sequence. In some embodiments, a replacementnucleic acid sequence is an ortholog of the replaced sequence. In someembodiments, a replacement nucleic acid sequence is or comprises a humannucleic acid sequence. In some embodiments, including where thereplacement nucleic acid sequence is or comprises a human nucleic acidsequence, the replaced nucleic acid sequence is or comprises a rodentsequence (e.g., a mouse or rat sequence). The nucleic acid sequence soplaced may include one or more regulatory sequences that are part ofsource nucleic acid sequence used to obtain the sequence so placed(e.g., promoters, enhancers, 5′- or 3′-untranslated regions, etc.). Forexample, in various embodiments, the replacement is a substitution of anendogenous sequence with a heterologous sequence that results in theproduction of a gene product from the nucleic acid sequence so placed(comprising the heterologous sequence), but not expression of theendogenous sequence; the replacement is of an endogenous genomicsequence with a nucleic acid sequence that encodes a polypeptide thathas a similar function as a polypeptide encoded by the endogenoussequence (e.g., the endogenous genomic sequence encodes a PD-1polypeptide, and the DNA fragment encodes one or more human PD-1polypeptides). In various embodiments, an endogenous gene or fragmentthereof is replaced with a corresponding human gene or fragment thereof.A corresponding human gene or fragment thereof is a human gene orfragment that is an ortholog of, or is substantially similar or the samein structure and/or function, as the endogenous gene or fragment thereofthat is replaced.

The phrase “Programmed cell death 1 protein” or “PD-1 protein” includesa type I transmembrane protein that belongs to the CD28/CTLA-4 family ofT cell regulators. The protein structure of a PD-1 protein includes anextracellular amino-terminal immunoglobulin V domain, a transmembranedomain and a carboxyl-terminal intracellular tail, which intracellulartail contains an immunoreceptor tyrosine-based inhibitory motif (ITIM)and an immunoreceptor tyrosine-based switch motif. PD-1 is expressed onthe cell surface and interacts with PD-L1 and PD-L2, members of the B7family immune-regulatory ligands (Collins, M. et al. (2005) Genome Biol.6:223). PD-1 is expressed in, inter alia, activated T cells, B cells,macrophages, monocytes, mast cells, and also in many tumors. PD-1 hasbeen shown to be involved in negative regulation of immune response and,in particular, negative regulation of T cell responses. By way ofillustration, nucleotide and amino acid sequences of mouse and humanPdcd1 genes, which encode PD-1 proteins, are provided in FIG. 8. Personsof skill upon reading this disclosure will recognize that one or moreendogenous Pdcd1 genes in a genome (or all) can be replaced by one ormore heterologous Pdcd1 genes (e.g., polymorphic variants, subtypes ormutants, genes from another species, humanized forms, etc.).

A “PD-1-expressing cell” includes a cell that expresses a PD-1 type Imembrane protein. In some embodiments, a PD-1-expressing cell expressesa PD-1 type I membrane protein on its surface. In some embodiments, aPD-1 protein is expressed on the surface of the cell in an amountsufficient to mediate cell-to-cell interactions. ExemplaryPD-1-expressing cells include B cells, macrophages and T cells.PD-1-expressing cells regulate various cellular processes via theinteraction of PD-1 expressed on the surface of immune cells (e.g., Tand B cells) and play a role in determining the differentiation and fateof such cells. In some embodiments, non-human animals of the presentinvention demonstrate regulation of various cellular processes (asdescribed herein) via humanized PD-1 proteins expressed on the surfaceof one more cells of the non-human animal. In some embodiments,non-human animals of the present invention demonstrate negativeregulation of signaling through T cell receptors (TCRs) via humanizedPD-1 proteins expressed on the surface of one or more cells of thenon-human animal. In some embodiments, non-human animals demonstratenegative regulation of immune responses via humanized PD-1 proteinsexpressed on the surface of one or more cells of the non-human animal.

The term “reference” includes a standard or control agent, cohort,individual, population, sample, sequence or value against which anagent, animal, cohort, individual, population, sample, sequence or valueof interest is compared. In some embodiments, a reference agent, cohort,individual, population, sample, sequence or value is tested and/ordetermined substantially simultaneously with the testing ordetermination of the agent, cohort, individual, population, sample,sequence or value of interest. In some embodiments, a reference agent,cohort, individual, population, sample, sequence or value is ahistorical reference, optionally embodied in a tangible medium. In someembodiments, a reference may refer to a control. As used herein, a“reference” may include a “reference animal”. A “reference animal” mayhave a modification as described herein, a modification that isdifferent as described herein or no modification (i.e., a wild-typeanimal). Typically, as would be understood by those skilled in the art,a reference agent, animal, cohort, individual, population, sample,sequence or value is determined or characterized under conditionscomparable to those utilized to determine or characterize the agent,animal (e.g., a mammal), cohort, individual, population, sample,sequence or value of interest.

The term “substantially” includes the qualitative condition ofexhibiting total or near-total extent or degree of a characteristic orproperty of interest. One of ordinary skill in the biological arts willunderstand that biological and chemical phenomena rarely, if ever, go tocompletion and/or proceed to completeness or achieve or avoid anabsolute result. The term “substantially” is therefore used herein tocapture the potential lack of completeness inherent in many biologicaland chemical phenomena.

The phrase “substantial homology” includes a comparison between aminoacid or nucleic acid sequences. As will be appreciated by those ofordinary skill in the art, two sequences are generally considered to be“substantially homologous” if they contain homologous residues incorresponding positions. Homologous residues may be identical residues.Alternatively, homologous residues may be non-identical residues willappropriately similar structural and/or functional characteristics. Forexample, as is well known by those of ordinary skill in the art, certainamino acids are typically classified as “hydrophobic” or “hydrophilic”amino acids, and/or as having “polar” or “non-polar” side chains.Substitution of one amino acid for another of the same type may often beconsidered a “homologous” substitution. Typical amino acidcategorizations are summarized in Table 1 and 2.

TABLE 1 Alanine Ala A Nonpolar Neutral 1.8 Arginine Arg R Polar Positive−4.5 Asparagine Asn N Polar Neutral −3.5 Aspartic acid Asp D PolarNegative −3.5 Cysteine Cys C Nonpolar Neutral 2.5 Glutamic acid Glu EPolar Negative −3.5 Glutamine Gln Q Polar Neutral −3.5 Glycine Gly GNonpolar Neutral −0.4 Histidine His H Polar Positive −3.2 Isoleucine IleI Nonpolar Neutral 4.5 Leucine Leu L Nonpolar Neutral 3.8 Lysine Lys KPolar Positive −3.9 Methionine Met M Nonpolar Neutral 1.9 PhenylalaninePhe F Nonpolar Neutral 2.8 Proline Pro P Nonpolar Neutral −1.6 SerineSer S Polar Neutral −0.8 Threonine Thr T Polar Neutral −0.7 TryptophanTrp W Nonpolar Neutral −0.9 Tyrosine Tyr Y Polar Neutral −1.3 Valine ValV Nonpolar Neutral 4.2

TABLE 2 Ambiguous Amino Acids 3-Letter 1-Letter Asparagine or asparticacid Asx B Glutamine or glutamic acid Glx Z Leucine or Isoleucine Xle JUnspecified or unknown amino acid Xaa X

As is well known in this art, amino acid or nucleic acid sequences maybe compared using any of a variety of algorithms, including thoseavailable in commercial computer programs such as BLASTN for nucleotidesequences and BLASTP, gapped BLAST, and PSI-BLAST for amino acidsequences. Exemplary such programs are described in Altschul et al.(1990) Basic local alignment search tool, J. Mol. Biol., 215(3):403-410; Altschul et al. (1997) Methods in Enzymology; Altschul et al.,“Gapped BLAST and PSI-BLAST: a new generation of protein database searchprograms”, Nucleic Acids Res. 25:3389-3402; Baxevanis et al. (1998)Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins,Wiley; and Misener et al. (eds.) (1999) Bioinformatics Methods andProtocols (Methods in Molecular Biology, Vol. 132), Humana Press. Inaddition to identifying homologous sequences, the programs mentionedabove typically provide an indication of the degree of homology. In someembodiments, two sequences are considered to be substantially homologousif at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more of their corresponding residues arehomologous over a relevant stretch of residues. In some embodiments, therelevant stretch is a complete sequence. In some embodiments, therelevant stretch is at least 9, 10, 11, 12, 13, 14, 15, 16, 17 or moreresidues. In some embodiments, the relevant stretch includes contiguousresidues along a complete sequence. In some embodiments, the relevantstretch includes discontinuous residues along a complete sequence. Insome embodiments, the relevant stretch is at least 10, 15, 20, 25, 30,35, 40, 45, 50, or more residues.

The phrase “substantial identity” includes a comparison between aminoacid or nucleic acid sequences. As will be appreciated by those ofordinary skill in the art, two sequences are generally considered to be“substantially identical” if they contain identical residues incorresponding positions. As is well known in this art, amino acid ornucleic acid sequences may be compared using any of a variety ofalgorithms, including those available in commercial computer programssuch as BLASTN for nucleotide sequences and BLASTP, gapped BLAST, andPSI-BLAST for amino acid sequences. Exemplary such programs aredescribed in Altschul et al. (1990) Basic local alignment search tool,J. Mol. Biol., 215(3): 403-410; Altschul et al., Methods in Enzymology;Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402; Baxevanis et al.(1998) Bioinformatics: A Practical Guide to the Analysis of Genes andProteins, Wiley; and Misener et al., (eds.) (1999) BioinformaticsMethods and Protocols (Methods in Molecular Biology, Vol. 132), HumanaPress. In addition to identifying identical sequences, the programsmentioned above typically provide an indication of the degree ofidentity. In some embodiments, two sequences are considered to besubstantially identical if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of theircorresponding residues are identical over a relevant stretch ofresidues. In some embodiments, the relevant stretch is a completesequence. In some embodiments, the relevant stretch is at least 10, 15,20, 25, 30, 35, 40, 45, 50, or more residues.

The phrase “targeting vector” or “targeting construct” includes apolynucleotide molecule that comprises a targeting region. A targetingregion comprises a sequence that is identical or substantially identicalto a sequence in a target cell, tissue or animal and provides forintegration of the targeting construct into a position within the genomeof the cell, tissue or animal via homologous recombination. Targetingregions that target using site-specific recombinase recognition sites(e.g., loxP or Frt sites) are also included. In some embodiments, atargeting construct of the present invention further comprises a nucleicacid sequence or gene of particular interest, a selectable marker,control and or regulatory sequences, and other nucleic acid sequencesthat allow for recombination mediated through exogenous addition ofproteins that aid in or facilitate recombination involving suchsequences. In some embodiments, a targeting construct of the presentinvention further comprises a gene of interest in whole or in part,wherein the gene of interest is a heterologous gene that encodes aprotein, in whole or in part, that has a similar function as a proteinencoded by an endogenous sequence. In some embodiments, a targetingconstruct of the present invention further comprises a humanized gene ofinterest, in whole or in part, wherein the humanized gene of interestencodes a protein, in whole or in part, that has a similar function as aprotein encoded by the endogenous sequence.

The phrase “therapeutically effective amount” includes an amount thatproduces the desired effect for which it is administered. In someembodiments, the term refers to an amount that is sufficient, whenadministered to a subject (e.g., an animal) suffering from orsusceptible to a disease, disorder, and/or condition in accordance witha therapeutic dosing regimen, to treat the disease, disorder, and/orcondition. In some embodiments, a therapeutically effective amount isone that reduces the incidence and/or severity of, and/or delays onsetof, one or more symptoms of the disease, disorder, and/or condition.Those of ordinary skill in the art will appreciate that the term“therapeutically effective amount” does not in fact require successfultreatment be achieved in a particular individual. Rather, atherapeutically effective amount may be that amount that provides aparticular desired pharmacological response in a significant number ofsubjects when administered to subjects in need of such treatment. Insome embodiments, reference to a therapeutically effective amount may bea reference to an amount as measured in one or more specific tissues(e.g., a tissue affected by the disease, disorder or condition) orfluids (e.g., blood, saliva, serum, sweat, tears, urine, etc.). Those ofordinary skill in the art will appreciate that, in some embodiments, atherapeutically effective amount of a particular agent or therapy may beformulated and/or administered in a single dose. In some embodiments, atherapeutically effective agent may be formulated and/or administered ina plurality of doses, for example, as part of a dosing regimen.

The term “treatment” (also “treat” or “treating”), in its broadest senseincludes any administration of a substance (e.g., provided compositions)that partially or completely alleviates, ameliorates, relives, inhibits,delays onset of, reduces severity of, and/or reduces incidence of one ormore symptoms, features, and/or causes of a particular disease,disorder, and/or condition. In some embodiments, such treatment may beadministered to a subject who does not exhibit signs of the relevantdisease, disorder and/or condition and/or of a subject who exhibits onlyearly signs of the disease, disorder, and/or condition. Alternatively oradditionally, in some embodiments, treatment may be administered to asubject who exhibits one or more established signs of the relevantdisease, disorder and/or condition. In some embodiments, treatment maybe of a subject who has been diagnosed as suffering from the relevantdisease, disorder, and/or condition. In some embodiments, treatment maybe of a subject known to have one or more susceptibility factors thatare statistically correlated with increased risk of development of therelevant disease, disorder, and/or condition.

The term “variant” includes an entity that shows significant structuralidentity with a reference entity, but differs structurally from thereference entity in the presence or level of one or more chemicalmoieties as compared with the reference entity. In many embodiments, a“variant” also differs functionally from its reference entity. Ingeneral, whether a particular entity is properly considered to be a“variant” of a reference entity is based on its degree of structuralidentity with the reference entity. As will be appreciated by thoseskilled in the art, any biological or chemical reference entity hascertain characteristic structural elements. A “variant”, by definition,is a distinct chemical entity that shares one or more suchcharacteristic structural elements. To give but a few examples, a smallmolecule may have a characteristic core structural element (e.g., amacrocycle core) and/or one or more characteristic pendent moieties sothat a variant of the small molecule is one that shares the corestructural element and the characteristic pendent moieties but differsin other pendent moieties and/or in types of bonds present (single vs.double, E vs. Z, etc.) within the core, a polypeptide may have acharacteristic sequence element comprised of a plurality of amino acidshaving designated positions relative to one another in linear orthree-dimensional space and/or contributing to a particular biologicalfunction, a nucleic acid may have a characteristic sequence elementcomprised of a plurality of nucleotide residues having designatedpositions relative to on another in linear or three-dimensional space.For example, a “variant polypeptide” may differ from a referencepolypeptide as a result of one or more differences in amino acidsequence and/or one or more differences in chemical moieties (e.g.,carbohydrates, lipids, etc.) covalently attached to the polypeptidebackbone. In some embodiments, a “variant polypeptide” shows an overallsequence identity with a reference polypeptide that is at least 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%.Alternatively or additionally, in some embodiments, a “variantpolypeptide” does not share at least one characteristic sequence elementwith a reference polypeptide. In some embodiments, the referencepolypeptide has one or more biological activities. In some embodiments,a “variant polypeptide” shares one or more of the biological activitiesof the reference polypeptide. In some embodiments, a “variantpolypeptide” lacks one or more of the biological activities of thereference polypeptide. In some embodiments, a “variant polypeptide”shows a reduced level of one or more biological activities as comparedwith the reference polypeptide. In many embodiments, a polypeptide ofinterest is considered to be a “variant” of a parent or referencepolypeptide if the polypeptide of interest has an amino acid sequencethat is identical to that of the parent but for a small number ofsequence alterations at particular positions. Typically, fewer than 20%,15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% of the residues in the variantare substituted as compared with the parent. In some embodiments, a“variant” has 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substituted residue ascompared with a parent. Often, a “variant” has a very small number(e.g., fewer than 5, 4, 3, 2, or 1) number of substituted functionalresidues (i.e., residues that participate in a particular biologicalactivity). Furthermore, a “variant” typically has not more than 5, 4, 3,2, or 1 additions or deletions, and often has no additions or deletions,as compared with the parent. Moreover, any additions or deletions aretypically fewer than about 25, about 20, about 19, about 18, about 17,about 16, about 15, about 14, about 13, about 10, about 9, about 8,about 7, about 6, and commonly are fewer than about 5, about 4, about 3,or about 2 residues. In some embodiments, the parent or referencepolypeptide is one found in nature. As will be understood by those ofordinary skill in the art, a plurality of variants of a particularpolypeptide of interest may commonly be found in nature, particularlywhen the polypeptide of interest is an infectious agent polypeptide.

The term “vector” includes a nucleic acid molecule capable oftransporting another nucleic acid to which it is associated. In someembodiment, vectors are capable of extra-chromosomal replication and/orexpression of nucleic acids to which they are linked in a host cell suchas a eukaryotic and/or prokaryotic cell. Vectors capable of directingthe expression of operatively linked genes are referred to herein as“expression vectors.”

The term “wild-type” includes an entity having a structure and/oractivity as found in nature in a “normal” (as contrasted with mutant,diseased, altered, etc.) state or context. Those of ordinary skill inthe art will appreciate that wild-type genes and polypeptides oftenexist in multiple different forms (e.g., alleles).

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

The present invention provides, among other things, improved and/orengineered non-human animals having humanized genetic material encodinga Programmed cell death 1 (Pdcd1) gene for determining the therapeuticefficacy of Pdcd1 modulators (e.g., an anti-PD-1 antibody) for thetreatment of cancer, and assays in T cell responses and signaltransduction. It is contemplated that such non-human animals provide animprovement in determining the therapeutic efficacy of PD-1 modulatorsand their potential for PD-1 blockade. Therefore, the present inventionis particularly useful for the development of anti-PD-1 therapies forthe treatment of various cancers, as well as for augmenting immuneresponses to treat and/or remove viral infection in non-human animals.In particular, the present invention encompasses the humanization of amurine Pdcd1 gene resulting in expression of a humanized PD-1 protein onthe surface of cells of the non-human animal. Such humanized PD-1proteins have the capacity to provide a source of human PD-1⁺ cells fordetermining the efficacy of anti-PD-1 therapeutics to promote anti-tumorimmune responses. In some embodiments, non-human animals of the presentinvention demonstrate augmented immune responses via blockade of PD-1signaling through the humanized PD-1 protein expressed on the surface ofcells of the non-human animal. In some embodiments, humanized PD-1proteins have a sequence corresponding to the N-terminal immunoglobulinV domain, in whole or in part, of a human PD-1 protein. In someembodiments, humanized PD-1 proteins have a sequence corresponding tothe intracellular tail of a murine PD-1 protein; in some embodiments, asequence corresponding to the transmembrane domain and intracellulartail of a murine PD-1 protein. In some embodiments, humanized PD-1proteins have a sequence corresponding to amino acid residues 21-170 (or26-169, 27-169, or 27-145, or 35-145) of a human PD-1 protein. In someembodiments, non-human animals of the present invention comprise anendogenous Pdcd1 gene that contains genetic material from the non-humananimal and a heterologous species (e.g., a human). In some embodiments,non-human animals of the present invention comprise a humanized Pdcd1gene, wherein the humanized Pdcd1 gene comprises exon 2 and exon 3, inwhole or in part, of a human PDCD1 gene. In some certain embodiments,non-human animals of the present invention comprise a humanized Pdcd1gene, wherein the humanized Pdcd1 gene comprises 883 bp of a human PDCD1gene corresponding to exon 2 and the first 71 bp of exon 3 (i.e.,encoding the stalk) of a human PDCD1 gene.

Various aspects of the invention are described in detail in thefollowing sections. The use of sections is not meant to limit theinvention. Each section can apply to any aspect of the invention. Inthis application, the use of “or” means “and/or” unless statedotherwise.

Programmed Cell Death 1 (Pdcd1) Gene

Pdcd1 (also referred to as CD279) was originally discovered as anupregulated gene in a T cell hybridoma that was undergoing apoptosis(Ishida, Y. et al. (1992) EMBO J. 11(11):3887-3895). The Pdcd1 geneconsists of 5 exons that encode PD-1, which is a type I membrane protein(referred to as PD-1) that includes an N-terminal immunoglobulin V (IgV)domain, a stalk (˜20 amino acids in length), a transmembrane domain, andan intracellular tail that contains both an immunoreceptor tyrosineinhibitory motif (ITIM) and an immunoreceptor tyrosine switch motif(ITSM). PD-1 is expressed on many cell types such as, for example, Bcells, dendritic cells, activated monocytes, natural killer (NK) cellsand activated T cells (Keir, M. E., et al. (2008) Annu. Rev. Immunol.26:677-704). Various splice variants of PD-1 have also been reported andvary based on which exon is lacking (Nielsen, C. et al. (2005) Cell.Immunol. 235:109-116). Indeed, certain splice variants have beenobserved as a causative factor in autoimmune diseases (Wan, B. et al.(2006) J. Immunol. 177(12):8844-8850). Further, Pdcd1-deficient micehave been reported to develop autoimmune conditions (Nishimura, H. etal. (1998) Intern. Immunol. 10(10):1563-1572; Nishimura, H. et al.(1999) Immunity 11:141-151; Nishimura, H. et al. (2001) Science291:319-322), which have lead the way to solidifying PD-1 as a negativeregulator of activated lymphocytes and serves to protect against thedevelopment of autoimmune disease. Interestingly, tumors have beendiscovered to use PD-1 signaling to evade surveillance by the immunesystem. Therefore, PD-1 and at least one of its ligands (i.e., PD-L1)are currently being explored as targets for cancer therapy by promotionof anti-tumor activity in tumor microenvironments via PD-1 blockade (seee.g., Pedoeem, A. et al. (2014) Clin. Immunol. 153:145-152; and Philips,G. K. and Atkins, M. (2014) Intern. Immunol. 8 pages).

A more thorough and detailed understanding of PD-1-mediated functionsand the PD-1 pathway is needed to develop practical targeted therapiesfor future cancer treatment.

Pdcd1 and PD-1 Sequences

Exemplary murine, human and humanized Pdcd1 and PD-1 sequences are setforth in FIG. 8. An exemplary human nucleic acid sequence forhumanization of a non-human Pdcd1 gene is also set forth in FIG. 8.

Humanized Pdcd1 Non-Human Animals

Non-human animals are provided that express humanized PD-1 proteins onthe surface of cells of the non-human animals resulting from a geneticmodification of an endogenous locus (e.g., a Pdcd1 locus) of thenon-human animal that encodes a PD-1 protein. Suitable examplesdescribed herein include rodents, in particular, mice.

A humanized Pdcd1 gene, in some embodiments, comprises genetic materialfrom a heterologous species (e.g., humans), wherein the humanized Pdcd1gene encodes a PD-1 protein that comprises the encoded portion of thegenetic material from the heterologous species. In some embodiments, ahumanized Pdcd1 gene of the present invention comprises genomic DNA of aheterologous species that encodes the extracellular portion of a PD-1protein that is expressed on the plasma membrane of a cell. Non-humananimals, embryos, cells and targeting constructs for making non-humananimals, non-human embryos, and cells containing said humanized Pdcd1gene are also provided.

In some embodiments, an endogenous Pdcd1 gene is deleted. In someembodiments, an endogenous Pdcd1 gene is altered, wherein a portion ofthe endogenous Pdcd1 gene is replaced with a heterologous sequence(e.g., a human PDCD1 sequence, in whole or in part). In someembodiments, all or substantially all of an endogenous Pdcd1 gene isreplaced with a heterologous gene (e.g., a human PDCD1 gene). In someembodiments, a portion of a heterologous Pdcd1 gene is inserted into anendogenous non-human Pdcd1 gene at an endogenous Pdcd1 locus. In someembodiments, the heterologous gene is a human gene. In some embodiments,the modification or humanization is made to one of the two copies of theendogenous Pdcd1 gene, giving rise to a non-human animal that isheterozygous with respect to the humanized Pdcd1 gene. In otherembodiments, a non-human animal is provided that is homozygous for ahumanized Pdcd1 gene.

In various aspects, a non-human animal contains a human PDCD1 gene, inwhole or in part, at an endogenous non-human Pdcd1 locus. Thus, suchnon-human animals can be described as having a heterologous Pdcd1 gene.The replaced, inserted, modified or altered Pdcd1 gene at the endogenousPdcd1 locus or a protein expressed from such gene can be detected usinga variety of methods including, for example, PCR, Western blot, Southernblot, restriction fragment length polymorphism (RFLP), or a gain or lossof allele assay. In some embodiments, the non-human animal isheterozygous with respect to the humanized Pdcd1 gene.

In various embodiments, a humanized Pdcd1 gene according to the presentinvention includes a Pdcd1 gene that has a second exon having a sequenceat least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to a secondexon that appears in a human PDCD1 gene of FIG. 8.

In various embodiments, a humanized Pdcd1 gene according to the presentinvention includes a Pdcd1 gene that has a second exon having a sequencethat is substantially identical to a second exon that appears in a humanPDCD1 gene of FIG. 8.

In various embodiments, a humanized Pdcd1 gene according to the presentinvention includes a Pdcd1 gene that has a second exon having a sequencethat is identical to a second exon that appears in a human PDCD1 gene ofFIG. 8.

In various embodiments, a humanized Pdcd1 gene according to the presentinvention includes a Pdcd1 gene that has a third exon having a sequenceat least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to a thirdexon that appears in a humanized Pdcd1 mRNA sequence of FIG. 8.

In various embodiments, a humanized Pdcd1 gene according to the presentinvention includes a Pdcd1 gene that has a third exon having a sequencethat is substantially identical to a third exon that appears in ahumanized Pdcd1 mRNA sequence of FIG. 8.

In various embodiments, a humanized Pdcd1 gene according to the presentinvention includes a Pdcd1 gene that has a third exon having a sequencethat is identical to a third exon that appears in a humanized Pdcd1 mRNAsequence of FIG. 8.

In various embodiments, a humanized Pdcd1 gene according to the presentinvention includes a Pdcd1 gene that comprises a sequence at least 50%(e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more) identical to SEQ ID NO:21 or SEQ IDNO:23.

In various embodiments, a humanized Pdcd1 gene according to the presentinvention includes a Pdcd1 gene that comprises a sequence that issubstantially identical to SEQ ID NO:21 or SEQ ID NO:23.

In various embodiments, a humanized Pdcd1 gene according to the presentinvention includes a Pdcd1 gene that comprises a sequence that isidentical to SEQ ID NO:21 or SEQ ID NO:23.

In various embodiments, a humanized Pdcd1 gene according to the presentinvention includes a Pdcd1 gene that has a second exon and a portion ofa third exon each having a sequence at least 50% (e.g., 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more) identical to a second exon and a portion of a third exonthat appear in a human PDCD1 gene of FIG. 8.

In various embodiments, a humanized Pdcd1 gene according to the presentinvention includes a Pdcd1 gene that has a first, fourth and fifth exoneach having a sequence at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more)identical to a first, fourth and fifth exon that appear in a mouse Pdcd1gene of FIG. 8.

In various embodiments, a humanized Pdcd1 gene according to the presentinvention includes a Pdcd1 gene that has a first, a portion of a third,a fourth and a fifth exon each having a sequence at least 50% (e.g.,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more) identical to a first, a portion of a third,a fourth and a fifth exon that appear in a mouse Pdcd1 gene of FIG. 8.

In various embodiments, a humanized Pdcd1 gene according to the presentinvention includes a Pdcd1 gene that has a 5′ untranslated region and a3′ untranslated region each having a sequence at least 50% (e.g., 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more) identical to a 5′ untranslated region and a 3′untranslated region that appear in a mouse Pdcd1 gene of FIG. 8.

In various embodiments, a humanized Pdcd1 gene according to the presentinvention includes a Pdcd1 gene that has a nucleotide coding sequence(e.g., a cDNA sequence) at least 50% (e.g., 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more)identical to a nucleotide coding sequence that appears in a humanizedPdcd1 nucleotide coding sequence of FIG. 8.

In various embodiments, a humanized Pdcd1 mRNA sequence according to thepresent invention comprises a sequence that is at least 50% (e.g., 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more) identical to a humanized mRNA sequence thatappears in FIG. 8.

In various embodiments, a humanized Pdcd1 gene according to the presentinvention encodes a PD-1 polypeptide having an amino acid sequence atleast 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to an amino acidsequence that appears in a PD-1 polypeptide sequence of FIG. 8.

In various embodiments, a humanized PD-1 protein produced by a non-humananimal of the present invention has an extracellular portion having anamino acid sequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more)identical to an extracellular portion of a human PD-1 protein thatappears in FIG. 8.

In various embodiments, a humanized PD-1 protein produced by a non-humananimal of the present invention has an extracellular portion, whichextracellular portion comprises an amino acid sequence that is at least50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more) identical to amino acid residues21-170 that appear in a human or humanized PD-1 protein of FIG. 8.

In various embodiments, a humanized PD-1 protein produced by a non-humananimal of the present invention has an extracellular portion, whichextracellular portion comprises an amino acid sequence that issubstantially identical to amino acid residues 21-170 that appear in ahuman or humanized PD-1 protein of FIG. 8.

In various embodiments, a humanized PD-1 protein produced by a non-humananimal of the present invention has an extracellular portion, whichextracellular portion comprises an amino acid sequence that is identicalto amino acid residues 21-170 that appear in a human or humanized PD-1protein of FIG. 8.

In various embodiments, a humanized PD-1 protein produced by a non-humananimal of the present invention has an extracellular portion, whichextracellular portion comprises an amino acid sequence that is at least50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more) identical to amino acid residues26-169 that appear in a human or humanized PD-1 protein of FIG. 8.

In various embodiments, a humanized PD-1 protein produced by a non-humananimal of the present invention has an extracellular portion, whichextracellular portion comprises an amino acid sequence that issubstantially identical to amino acid residues 26-169 that appear in ahuman or humanized PD-1 protein of FIG. 8.

In various embodiments, a humanized PD-1 protein produced by a non-humananimal of the present invention has an extracellular portion, whichextracellular portion comprises an amino acid sequence that is identicalto amino acid residues 26-169 that appear in a human or humanized PD-1protein of FIG. 8.

In various embodiments, a humanized PD-1 protein produced by a non-humananimal of the present invention has an extracellular portion, whichextracellular portion comprises an amino acid sequence that is at least50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more) identical to amino acid residues27-169 that appear in a human or humanized PD-1 protein of FIG. 8.

In various embodiments, a humanized PD-1 protein produced by a non-humananimal of the present invention has an extracellular portion, whichextracellular portion comprises an amino acid sequence that issubstantially identical to amino acid residues 27-169 that appear in ahuman or humanized PD-1 protein of FIG. 8.

In various embodiments, a humanized PD-1 protein produced by a non-humananimal of the present invention has an extracellular portion, whichextracellular portion comprises an amino acid sequence that is identicalto amino acid residues 27-169 that appear in a human or humanized PD-1protein of FIG. 8.

In various embodiments, a humanized PD-1 protein produced by a non-humananimal of the present invention has an extracellular portion, whichextracellular portion comprises an amino acid sequence that is at least50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more) identical to amino acid residues27-145 that appear in a human or humanized PD-1 protein of FIG. 8.

In various embodiments, a humanized PD-1 protein produced by a non-humananimal of the present invention has an extracellular portion, whichextracellular portion comprises an amino acid sequence that issubstantially identical to amino acid residues 27-145 that appear in ahuman or humanized PD-1 protein of FIG. 8.

In various embodiments, a humanized PD-1 protein produced by a non-humananimal of the present invention has an extracellular portion, whichextracellular portion comprises an amino acid sequence that is identicalto amino acid residues 27-145 that appear in a human or humanized PD-1protein of FIG. 8.

In various embodiments, a humanized PD-1 protein produced by a non-humananimal of the present invention has an extracellular portion, whichextracellular portion comprises an amino acid sequence that is at least50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more) identical to amino acid residues35-145 that appear in a human or humanized PD-1 protein of FIG. 8.

In various embodiments, a humanized PD-1 protein produced by a non-humananimal of the present invention has an extracellular portion, whichextracellular portion comprises an amino acid sequence that issubstantially identical to amino acid residues 35-145 that appear in ahuman or humanized PD-1 protein of FIG. 8.

In various embodiments, a humanized PD-1 protein produced by a non-humananimal of the present invention has an extracellular portion, whichextracellular portion comprises an amino acid sequence that is identicalto amino acid residues 35-145 that appear in a human or humanized PD-1protein of FIG. 8.

In various embodiments, a humanized PD-1 protein produced by a non-humananimal of the present invention has an N-terminal immunoglobulin Vdomain having an amino acid sequence that is at least 50% (e.g., 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more) identical to an N-terminal immunoglobulin Vdomain of a human or humanized PD-1 protein that appears in FIG. 8.

In various embodiments, a humanized PD-1 protein produced by a non-humananimal of the present invention has an N-terminal immunoglobulin Vdomain having an amino acid sequence that is substantially identical toan N-terminal immunoglobulin V domain that appears in a human orhumanized PD-1 protein of FIG. 8.

In various embodiments, a humanized PD-1 protein produced by a non-humananimal of the present invention has an N-terminal immunoglobulin Vdomain having an amino acid sequence that is identical to an N-terminalimmunoglobulin V domain that appears in a human or humanized PD-1protein of FIG. 8.

In various embodiments, a humanized PD-1 protein produced by a non-humananimal of the present invention has a transmembrane domain having asequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identicalto a transmembrane domain of a mouse PD-1 protein that appears in FIG.8.

In various embodiments, a humanized PD-1 protein produced by a non-humananimal of the present invention has an intracellular tail having asequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identicalto an intracellular tail of a mouse PD-1 protein that appears in FIG. 8.

In various embodiments, a humanized PD-1 protein produced by a non-humananimal of the present invention has an amino acid sequence that is atleast 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to amino acidresidues 27-169 (or 26-169) that appear in a human PD-1 protein of FIG.8.

In various embodiments, a humanized PD-1 protein produced by a non-humananimal of the present invention has an amino acid sequence that issubstantially identical to amino acid residues 27-169 (or 26-169) thatappear in a human PD-1 protein of FIG. 8.

In various embodiments, a humanized PD-1 protein produced by a non-humananimal of the present invention has an amino acid sequence that isidentical to amino acid residues 27-169 (or 26-169) that appear in ahuman PD-1 protein of FIG. 8.

In various embodiments, a humanized PD-1 protein produced by a non-humananimal of the present invention has an amino acid sequence that is atleast 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to an amino acidsequence of a humanized PD-1 protein that appears in FIG. 8.

In various embodiments, a humanized PD-1 protein produced by a non-humananimal of the present invention has an amino acid sequence that issubstantially identical to an amino acid sequence of a humanized PD-1protein that appears in FIG. 8.

In various embodiments, a humanized PD-1 protein produced by a non-humananimal of the present invention has an amino acid sequence that isidentical to an amino acid sequence of a humanized PD-1 protein thatappears in FIG. 8.

Compositions and methods for making non-human animals that express ahumanized PD-1 protein, including specific polymorphic forms, allelicvariants (e.g., single amino acid differences) or alternatively splicedisoforms, are provided, including compositions and methods for makingnon-human animals that express such proteins from a human promoter and ahuman regulatory sequence. In some embodiments, compositions and methodsfor making non-human animals that express such proteins from anendogenous promoter and an endogenous regulatory sequence are alsoprovided. In some certain embodiments, endogenous promoters andendogenous regulatory sequences are endogenous rodent promoters andendogenous rodent regulatory sequences. The methods include insertingthe genetic material encoding a human PD-1 protein in whole or in partat a precise location in the genome of a non-human animal thatcorresponds to an endogenous Pdcd1 gene thereby creating a humanizedPdcd1 gene that expresses a PD-1 protein that is human in whole or inpart. In some embodiments, the methods include inserting genomic DNAcorresponding to exon 2 and exon 3, in whole or in part, of a humanPDCD1 gene into an endogenous Pdcd1 gene of the non-human animal therebycreating a humanized gene that encodes a PD-1 protein that contains ahuman portion containing amino acids encoded by the inserted exons.

Where appropriate, the coding region of the genetic material orpolynucleotide sequence(s) encoding a human (or humanized) PD-1 proteinin whole or in part may be modified to include codons that are optimizedfor expression from cells in the non-human animal (e.g., see U.S. Pat.Nos. 5,670,356 and 5,874,304). Codon optimized sequences are syntheticsequences, and preferably encode the identical polypeptide (or abiologically active fragment of a full length polypeptide which hassubstantially the same activity as the full length polypeptide) encodedby the non-codon optimized parent polynucleotide. In some embodiments,the coding region of the genetic material encoding a human (orhumanized) PD-1 protein, in whole or in part, may include an alteredsequence to optimize codon usage for a particular cell type (e.g., arodent cell). For example, the codons of the genomic DNA correspondingto exon 2 and a portion of exon 3 (e.g., 71 bp) of a human PDCD1 gene tobe inserted into an endogenous Pdcd1 gene of a non-human animal (e.g., arodent) may be optimized for expression in a cell of the non-humananimal. Such a sequence may be described as a codon-optimized sequence.

A humanized Pdcd1 gene approach employs a relatively minimalmodification of the endogenous gene and results in natural PD-1-mediatedsignal transduction in the non-human animal, in various embodiments,because the genomic sequence of the Pdcd1 sequences are modified in asingle fragment and therefore retain normal functionality by includingnecessary regulatory sequences. Thus, in such embodiments, the Pdcd1gene modification does not affect other surrounding genes or otherendogenous Pdcd1-interacting genes (e.g., PD-L1, PD-L2, etc.). Further,in various embodiments, the modification does not affect the assembly ofa functional PD-1 transmembrane protein on the cell membrane andmaintains normal effector functions via binding and subsequent signaltransduction through the cytoplasmic portion of the protein which isunaffected by the modification.

A schematic illustration (not to scale) of the genomic organization ofan endogenous murine Pdcd1 gene and a human PDCD1 gene is provided inFIG. 1. An exemplary method for humanizing an endogenous murine Pdcd1gene using a genomic fragment containing exon 2 and a portion of exon 3of a human PDCD1 gene is provided in FIG. 2. As illustrated, an 883 bpgenomic DNA fragment containing exon 2 and a portion of exon 3 (e.g.,the first 71 bp) of a human PDCD1 gene is inserted into the place of a900 bp sequence of an endogenous murine Pdcd1 gene locus by a targetingconstruct. The 883 bp human DNA fragment may be cloned directly fromhuman DNA or synthesized from a source sequence (e.g., Genbank accessionno. NM_005018.2). This genomic DNA includes the portion of the gene thatencodes substantially all of the extracellular portion (e.g., amino acidresidues 27-169 or 26-169) of a human PD-1 protein responsible forligand binding.

A non-human animal (e.g., a mouse) having a humanized Pdcd1 gene at theendogenous Pdcd1 locus can be made by any method known in the art. Forexample, a targeting vector can be made that introduces a human Pdcd1gene in whole or in part with a selectable marker gene. FIG. 2illustrates a targeting vector that contains an endogenous Pdcd1 locusof a mouse genome comprising an insertion of an 883 bp human DNAfragment that includes exon 2 and the first 71 bp of exon 3 of a humanPDCD1 gene. As illustrated, the targeting construct contains a 5′homology arm containing sequence upstream of exon 2 of an endogenousmurine Pdcd1 gene (˜61.7 Kb), followed by a drug selection cassette(e.g., a neomycin resistance gene flanked on both sides by loxPsequences; ˜5 Kb), a genomic DNA fragment containing exon 2 and thefirst 71 bp of exon 3 of a human Pdcd1 gene (883 bp), and a 3′ homologyarm containing the remaining sequence of an endogenous murine exon 3(i.e., portion which encodes a transmembrane portion of a PD-1 protein),exon 4 and exon 5 of an endogenous murine Pdcd1 gene (˜84 Kb). Thetargeting construct contains a self-deleting drug selection cassette(e.g., a neomycin resistance gene flanked by loxP sequences; see U.S.Pat. Nos. 8,697,851, 8,518,392 and 8,354,389, all of which are hereinincorporated by reference). Upon electroporation in embryonic stemcells, a modified endogenous Pdcd1 gene is created that exchanges 900 bpof an endogenous wild-type Pdcd1 gene with 883 bp of a human PDCD1 gene(i.e., exon 2 and the first 71 bp of exon 3), which is contained in thetargeting vector. A humanized Pdcd1 gene is created resulting in a cellor non-human animal that expresses a humanized PD-1 protein thatcontains amino acids encoded by the 883 bp human DNA fragment (i.e.,exon 2 and 71 bp of exon 3 of a human PDCD1 gene). The drug selectioncassette is removed in a development-dependent manner, i.e., progenyderived from mice whose germ line cells containing the humanized Pdcd1gene described above will shed the selectable marker from differentiatedcells during development (see bottom of FIG. 2).

Although embodiments employing a humanized Pdcd1 gene in a mouse (i.e.,a mouse with a Pdcd1 gene that encodes a PD-1 protein that includes ahuman portion and a mouse portion) are extensively discussed herein,other non-human animals that comprise a humanized Pdcd1 gene are alsoprovided. In some embodiments, such non-human animals comprise ahumanized Pdcd1 gene operably linked to a rodent Pdcd1 promoter. In someembodiments, such non-human animals comprise a humanized Pdcd1 geneoperably linked to an endogenous Pdcd1 promoter; in some embodiments, anendogenous rodent Pdcd1 promoter. In some embodiments, such non-humananimals express a humanized PD-1 protein from an endogenous locus,wherein the humanized PD-1 protein comprises amino acid residues 21-170(or 26-169, or 27-169, 27-145 or 35-145) of a human PD-1 protein. Suchnon-human animals include any of those which can be genetically modifiedto express a PD-1 protein as disclosed herein, including, e.g., mammals,e.g., mouse, rat, rabbit, pig, bovine (e.g., cow, bull, buffalo), deer,sheep, goat, chicken, cat, dog, ferret, primate (e.g., marmoset, rhesusmonkey), etc. For example, for those non-human animals for whichsuitable genetically modifiable ES cells are not readily available,other methods are employed to make a non-human animal comprising thegenetic modification. Such methods include, e.g., modifying a non-EScell genome (e.g., a fibroblast or an induced pluripotent cell) andemploying somatic cell nuclear transfer (SCNT) to transfer thegenetically modified genome to a suitable cell, e.g., an enucleatedoocyte, and gestating the modified cell (e.g., the modified oocyte) in anon-human animal under suitable conditions to form an embryo.

Methods for modifying a non-human animal genome (e.g., a pig, cow,rodent, chicken, etc. genome) include, e.g., employing a zinc fingernuclease (ZFN) or a transcription activator-like effector nuclease(TALEN) to modify a genome to include a humanized Pdcd1 gene.

In some embodiments, a non-human animal of the present invention is amammal. In some embodiments, a non-human animal of the present inventionis a small mammal, e.g., of the superfamily Dipodoidea or Muroidea. Insome embodiments, a genetically modified animal of the present inventionis a rodent. In some embodiments, a rodent of the present invention isselected from a mouse, a rat, and a hamster. In some embodiments, arodent of the present invention is selected from the superfamilyMuroidea. In some embodiments, a genetically modified animal of thepresent invention is from a family selected from Calomyscidae (e.g.,mouse-like hamsters), Cricetidae (e.g., hamster, New World rats andmice, voles), Muridae (true mice and rats, gerbils, spiny mice, crestedrats), Nesomyidae (climbing mice, rock mice, white-tailed rats, Malagasyrats and mice), Platacanthomyidae (e.g., spiny dormice), and Spalacidae(e.g., mole rates, bamboo rats, and zokors). In some certainembodiments, a genetically modified rodent of the present invention isselected from a true mouse or rat (family Muridae), a gerbil, a spinymouse, and a crested rat. In some certain embodiments, a geneticallymodified mouse of the present invention is from a member of the familyMuridae. In some embodiment, a non-human animal of the present inventionis a rodent. In some certain embodiments, a rodent of the presentinvention is selected from a mouse and a rat. In some embodiments, anon-human animal of the present invention is a mouse.

In some embodiments, a non-human animal of the present invention is arodent that is a mouse of a C57BL strain selected from C57BL/A,C57BL/An, C57BL/GrFa, C57BL/KaLwN, C57BL/6, C57BL/6J, C57BL/6ByJ,C57BL/6NJ, C57BL/10, C57BL/10ScSn, C57BL/10Cr, and C57BL/Ola. In somecertain embodiments, a mouse of the present invention is a 129 strainselected from the group consisting of a strain that is 129P1, 129P2,129P3, 129X1, 129S1 (e.g., 129S1/SV, 129S1/SvIm), 129S2, 129S4, 129S5,129S9/SvEvH, 129/SvJae, 129S6 (129/SvEvTac), 129S7, 129S8, 129T1, 129T2(see, e.g., Festing et al., 1999, Mammalian Genome 10:836; Auerbach, W.et al., 2000, Biotechniques 29(5):1024-1028, 1030, 1032). In somecertain embodiments, a genetically modified mouse of the presentinvention is a mix of an aforementioned 129 strain and an aforementionedC57BL/6 strain. In some certain embodiments, a mouse of the presentinvention is a mix of aforementioned 129 strains, or a mix ofaforementioned BL/6 strains. In some certain embodiments, a 129 strainof the mix as described herein is a 129S6 (129/SvEvTac) strain. In someembodiments, a mouse of the present invention is a BALB strain, e.g.,BALB/c strain. In some embodiments, a mouse of the present invention isa mix of a BALB strain and another aforementioned strain.

In some embodiments, a non-human animal of the present invention is arat. In some certain embodiments, a rat of the present invention isselected from a Wistar rat, an LEA strain, a Sprague Dawley strain, aFischer strain, F344, F6, and Dark Agouti. In some certain embodiments,a rat strain as described herein is a mix of two or more strainsselected from the group consisting of Wistar, LEA, Sprague Dawley,Fischer, F344, F6, and Dark Agouti.

Methods Employing Non-Human Animals Having Humanized Pdcd1 Genes

Investigation into PD-1 function has employed the use of various Pdcd1mutant and transgenic non-human animals (e.g., see Nishimura, H. et al.(1998) Intern. Immunol. 10(10):1563-1572; Nishimura, H. et al. (1999)Immunity 11:141-151; Nishimura, H. et al. (2001) Science 291:319-322;Iwai, Y. et al. (2004) Intern. Immunol. 17(2):133-144; Keir, M. E. etal. (2005) J. Immunol. 175:7372-7379; Keir, M. E. et al. (2007) J.Immunol. 179:5064-5070; Carter, L. L. et al. (2007) J. Neuroimmunol.182:124-134; Chen, L. et al. (2007) Europ. Soc. Organ Transplant.21:21-29; Okazaki, T. et al. (2011) J. Exp. Med. 208(2):395-407; U.S.Pat. No. 7,414,171; and European Patent No. 1 334 659 B1; whichreferences are herein incorporated by reference). Such mutant andtransgenic animals have been useful in determining the molecular aspectsof PD-1 expression, function and regulation of various cellularprocesses. However, they are not without limitation. For example,PD-1-deficient mice generated by knock-in of a human PD-1 cDNA into exon1 of a mouse Pdcd1 gene did not express human PD-1 even afterstimulation with PMA (Carter, L. L. et al., supra). Further,considerable phenotypic differences among PD-1 mutant animals indifferent genetic backgrounds has complicated investigation, especiallywhen attempting to assign various functions and/or regulatory activitiesto PD-1. Still, other transgenic animals have been created thatoverexpress PD-1 (Chen, L. et al., supra). Such animals have displayeddifferent expression patterns of the transgene, which can reasonably beattributed to construct design. Further, due to the use of the samesource genetic material (i.e., mouse), PD-1 overexpression may havecorresponded to endogenous PD-1 rather than transgenic PD-1 due topossible position effects of the transgene. While PD-1 transgenic micehave proved useful in elucidating some PD-1-mediated biologicalfunction, they have demonstrated variability in the results obtained,which are based, at least in part, from the different approachesemployed to make them. Therefore, current in vivo systems exploitingPD-1-mediated biology are incomplete. The molecular aspects ofPD-1-mediated biological function and signaling pathways has not beenexploited in transgenic mice to its fullest potential.

Non-human animals of the present invention provide an improved in vivosystem and source of biological materials (e.g., cells) expressing human(or humanized) PD-1 that are useful for a variety of assays. In variousembodiments, non-human animals of the present invention are used todevelop therapeutics that target PD-1 and/or modulate PD-1 signaling(e.g., interfering with interactions with PD-L1 and/or PD-L2). Invarious embodiments, non-human animals of the present invention are usedto identify, screen and/or develop candidate therapeutics (e.g.,antibodies) that bind human PD-1. In various embodiments, non-humananimals of the present invention are used to screen and developcandidate therapeutics (e.g., antibodies) that block interaction ofhuman PD-1 with human PD-L1 and/or human PD-L2. In various embodiments,non-human animals of the present invention are used to determine thebinding profile of antagonists and/or agonists of a humanized PD-1 onthe surface of a cell of a non-human animal as described herein; in someembodiments, non-human animals of the present invention are used todetermine the epitope or epitopes of one or more candidate therapeuticantibodies that bind human PD-1.

In various embodiments, non-human animals of the present invention areused to determine the pharmacokinetic profiles of anti-PD-1 antibodies.In various embodiments, one or more non-human animals of the presentinvention and one or more control or reference non-human animals areeach exposed to one or more candidate therapeutic anti-PD-1 antibodiesat various doses (e.g., 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/mg, 7.5 mg/kg, 10 mg/kg,15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 40 mg/kg, or 50 mg/kg or more).Candidate therapeutic antibodies may be dosed via any desired route ofadministration including parenteral and non-parenteral routes ofadministration. Parenteral routes include, e.g., intravenous,intraarterial, intraportal, intramuscular, subcutaneous,intraperitoneal, intraspinal, intrathecal, intracerebro ventricular,intracranial, intrapleural or other routes of injection. Non-parenteralroutes include, e.g., oral, nasal, transdermal, pulmonary, rectal,buccal, vaginal, ocular. Administration may also be by continuousinfusion, local administration, sustained release from implants (gels,membranes or the like), and/or intravenous injection. Blood is isolatedfrom non-human animals (humanized and control) at various time points(e.g., 0 hr, 6 hr, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7days, 8 days, 9 days, 10 days, 11 days, or up to 30 or more days).Various assays may be performed to determine the pharmacokineticprofiles of administered candidate therapeutic antibodies using samplesobtained from non-human animals as described herein including, but notlimited to, total IgG, anti-therapeutic antibody response,agglutination, etc.

In various embodiments, non-human animals of the present invention areused to measure the therapeutic effect of blocking or modulating PD-1signaling and the effect on gene expression as a result of cellularchanges. In various embodiments, a non-human animal of the presentinvention or cells isolated therefrom are exposed to a candidatetherapeutic that binds a humanized PD-1 protein (or a human portion of aPD-1 protein) on the surface of a cell of the non-human animal and,after a subsequent period of time, analyzed for effects onPD-1-dependent processes, for example, adhesion, apoptosis, cytokineproduction, inflammation, proliferation, self-tolerance and viralinfection (or responses).

Non-human animals of the present invention express humanized PD-1protein, thus cells, cell lines, and cell cultures can be generated toserve as a source of humanized PD-1 for use in binding and functionalassays, e.g., to assay for binding or function of a PD-1 antagonist oragonist, particularly where the antagonist or agonist is specific for ahuman PD-1 sequence or epitope or, alternatively, specific for a humanPD-1 sequence or epitope that associates with PD-L1 and/or PD-L2. Invarious embodiments, PD-1 epitopes bound by candidate therapeuticantibodies can be determined using cells isolated from non-human animalsof the present invention. In various embodiments, a humanized PD-1protein expressed by a non-human animal as described herein may comprisea variant amino acid sequence. Variant human PD-1 proteins (e.g.,polymorphisms) associated with autoimmune and infectious diseases havebeen reported (e.g., see Lee, Y. H. et al. (2014) Z. Rheumatol. PMID:24942602; Mansur, A. et al. (2014) J. Investig. Med. 62(3):638-643;Nasi, M. et al. (2013) Intern. J. Infect. Dis. 17:e845-e850; Piskin, I.E. et al. (2013) Neuropediatrics 44(4):187-190; Carter, L. L. et al.(2007) J. Neuroimmunol. 182(1-2):124-134; Wan, B. et al. (2006) J.Immunol. 177(12):8844-8850). Exemplary human PD-1 variants include thoselisted in the SNP GeneView webpage from NCBI and are summarized in Table3. In various embodiments, non-human animals of the present inventionexpress a humanized PD-1 protein variant. In various embodiments, thevariant is polymorphic at an amino acid position associated with ligandbinding. In various embodiments, non-human animals of the presentinvention are used to determine the effect of ligand binding throughinteraction with a polymorphic variant of human PD-1. In some certainembodiments, non-human animals of the present invention express a humanPD-1 variant that appears in Table 3.

TABLE 3 Chromosome mRNA Amino Amino acid position position Variant IDNo. Allele acid Codon position position 241851110 883 rs372765600 A Gln[Q] 2 272 G Arg [R] 2 272 241851118 875 rs368411538 T Asp [D] 3 269 CAsp [D] 3 269 241851121 872 rs2227981 A Ala [A] 3 268 C Ala [A] 3 268 GAla [A] 3 268 T Ala [A] 3 268 241851135 858 rs146642159 T Cys [C] 1 264C Arg [R] 1 264 241851138 855 rs143359677 A Thr [T] 1 263 G Ala [A] 1263 241851160 833 rs141228784 T Ser [S] 3 255 C Ser [S] 3 255 241851163830 rs200434733 C Pro [P] 3 254 T Pro [P] 3 254 241851171 822rs201961957 A Ile [I] 1 252 G Val [V] 1 252 241851188 805 rs201540918 TMet [M] 2 246 C Thr [T] 2 246 241851190 803 rs201481671 A Gln [Q] 3 245G Gln [Q] 3 245 241851210 783 rs137861407 A Met [M] 1 239 G Val [V] 1239 241851220 773 rs370462869 A Pro [P] 3 235 G Pro [P] 3 235 241851237756 rs147213978 C Arg [R] 1 230 T Trp [W] 1 230 241851264 729rs373940258 A Met [M] 1 221 G Val [V] 1 221 241851274 719 rs373831349 GPro [P] 3 217 T Pro [P] 3 217 241851279 714 rs376257658 A Met [M] 1 216G Val [V] 1 216 241851281 712 rs2227982 T Val [V] 2 215 C Ala [A] 2 215241851954 690 rs148456597 A Thr [T] 1 208 C Pro [P] 1 208 241851961 683rs146821282 G Thr [T] 3 205 T Thr [T] 3 205 C Thr [T] 3 205 241852204654 rs144217487 A Thr [T] 1 196 G Ala [A] 1 196 241852205 653rs141119263 T Ala [A] 3 195 C Ala [A] 3 195 241852209 649 rs200312345 AGln [Q] 2 194 G Arg [R] 2 194 241852258 600 rs55667829 T Leu [L] 1 178 CLeu [L] 1 178 241852271 587 rs377191240 T Val [V] 3 173 C Val [V] 3 173241852310 548 rs370660750 G Pro [P] 3 160 C Pro [P] 3 160 241852644 481rs138031190 A Gln [Q] 2 138 T Leu [L] 2 138 241852658 467 rs374762232 AGln [Q] 3 133 G Gln [Q] 3 133 241852661 464 rs41400345 A Ala [A] 3 132 GAla [A] 3 132 241852691 434 rs367833850 T Leu [L] 3 122 C Leu [L] 3 122241852697 428 rs186074812 T Thr [T] 3 120 C Thr [T] 3 120 241852715 410rs141299049 A Arg [R] 3 114 G Arg [R] 3 114 241852716 409 rs55679128 AGln [Q] 2 114 G Arg [R] 2 114 241852720 405 rs200323895 A Thr [T] 1 113G Ala [A] 1 113 241852729 396 rs190602950 A Met [M] 1 110 G Val [V] 1110 241852730 395 rs370268595 T Ser [S] 3 109 C Ser [S] 3 109 241852743382 rs368009835 G Gly [G] 2 105 A Asp [D] 2 105 241852746 379rs138016578 A His [H] 2 104 G Arg [R] 2 104 241852750 375 rs56124337 AArg [R] 1 103 G Gly [G] 1 103 241852751 374 rs55637807 T Asn [N] 3 102 CAsn [N] 3 102 241852755 370 rs371902970 T Leu [L] 2 101 C Pro [P] 2 101241852788 337 rs144257658 T Val [V] 2 90 G Gly [G] 2 90 241852808 317rs55804130 T Pro [P] 3 83 C Pro [P] 3 83 241852817 308 rs373755187 A Ala[A] 3 80 T Ala [A] 3 80 C Ala [A] 3 80 241852860 265 rs28615468 C Thr[T] 2 66 A Asn [N] 2 66 241852866 259 rs142434414 G Gly [G] 2 64 T Val[V] 2 64 241852877 248 rs181904226 A Ser [S] 3 60 G Ser [S] 3 60241852892 233 rs55993679 T Ser [S] 3 55 C Ser [S] 3 55 241852904 221rs373582646 G Thr [T] 3 51 C Thr [T] 3 51 241852910 215 rs141718335 TAsn [N] 3 49 C Asn [N] 3 49 241852922 203 rs374726495 T Thr [T] 3 45 CThr [T] 3 45 241852928 197 rs147586902 C Val [V] 3 43 G Val [V] 3 43241852930 195 rs368829632 A Met [M] 1 43 G Val [V] 1 43 241852951 174rs373081859 G Ala [A] 1 36 A Thr [T] 1 36 241852952 173 rs41444844 G Pro[P] 3 35 C Pro [P] 3 35 241852974 151 rs56234260 T Leu [L] 2 28 C Pro[P] 2 28 241858780 127 rs368550965 A Gln [Q] 2 20 G Arg [R] 2 20241858800 107 rs370111035 A Ala [A] 3 13 G Ala [A] 3 13 241858808 99rs142544044 A Ile [I] 1 11 G Val [V] 1 11

Cells from non-human animals of the present invention can be isolatedand used on an ad hoc basis, or can be maintained in culture for manygenerations. In various embodiments, cells from a non-human animal ofthe present invention are immortalized (e.g., via use of a virus) andmaintained in culture indefinitely (e.g., in serial cultures).

In various embodiments, cells and/or non-human animals of the presentinvention are used in various immunization regimens to determine thePD-1-mediated functions in the immune response to an antigen. In someembodiments, candidate therapeutics that bind to, or block one or morefunctions of, human (or humanized) PD-1 are characterized in a non-humananimal of the present invention. Suitable measurements include variouscellular assays, proliferation assays, serum immunoglobulin analysis(e.g., antibody titer), cytotoxicity assays, characterization ofligand-receptor interactions (e.g., immunoprecipitation assays). In someembodiments, non-human animals of the present invention are used tocharacterize the PD-1-mediated functions regulating an immune responseto an antigen. In some embodiments, the antigen is associated with anautoimmune disease, disorder or condition. In some embodiments, theantigen is associated with an inflammatory disease, disorder orcondition. In some embodiments, the antigen is a test antigen (e.g.,ovalbumin or OVA). In some embodiments, the antigen is a targetassociated with a disease or condition suffered by one or more humanpatients in need of treatment.

In various embodiments, non-human animals of the present invention areused in serum assays for determining titers of autoantibody productionfor testing the pharmaco-toxicological aspects of candidate therapeuticsthat target human PD-1. In some embodiments, autoantibody production innon-human animals of the present invention results from one or moreautoimmune diseases, disorders or conditions induced in the non-humananimal.

In various embodiments, non-human animals of the present invention areused for challenge with one or more antigens to determine thetherapeutic potential of compounds or biological agents to modulatePD-1-dependent regulation of an immune response, including but notlimited to, the specific T cell-dependent and B cell-dependent responsesto a given antigen.

In various embodiments, cells and/or non-human animals of the presentinvention are used in a survival and/or proliferation assay (e.g.,employing B or T cells) to screen and develop candidate therapeuticsthat modulate human PD-1 signaling Activation or loss of PD-1 plays animportant role in the regulation of T cell responses, and regulation ofself-tolerance by PD-1 may result from the activation of specificepitopes of the extracellular domain of PD-1, therefore, candidate PD-1modulators (e.g., antagonists or agonists) may be identified,characterized and developed using cells of non-human animals of thepresent invention and/or a non-human animal as described herein. In someembodiments, cells and/or non-human animals of the present invention areused in survival or death assay(s) to determine the effect onproliferation or apoptosis of a specific cell(s) (e.g., cancer cells) inthe presence and absence of PD-1.

In various embodiments, cells and/or non-human animals of the presentinvention are used in xenotransplantation of heterologous (e.g., human)cells or tissue to determine the PD-1-mediated functions in thephysiological (e.g., immune) response to the transplanted human cells ortissue. In some embodiments, candidate therapeutics that bind, or blockone or more functions of, human PD-1 are characterized in a non-humananimal of the present invention. Suitable measurements include variouscellular assays, proliferation assays, serum immunoglobulin analysis(e.g., antibody titer), cytotoxicity assays, and characterization ofligand-receptor interactions (immunoprecipitation assays). In someembodiments, non-human animals of the present invention are used tocharacterize the PD-1-mediated functions regulating an immune responseto an antigen. In some embodiments, the antigen is associated with aneoplasm. In some embodiments, the antigen is associated with anautoimmune disease, disorder or condition. In some embodiments, theantigen is associated with an inflammatory disease, disorder orcondition. In some embodiments, the antigen is a target associated witha disease or condition suffered by one or more human patients in need oftreatment.

In various embodiments, non-human animals of the present invention areused in transplantation or adoptive transfer experiments to determinethe therapeutic potential of compounds or biological agents to modulatePD-1-dependent regulation of new lymphocytes and their immune function.In various embodiments, non-human animals of the present invention aretransplanted with human T cells; in some embodiments, naïve T cells; insome embodiments, activated T cells.

In various embodiments, cells of non-human animals of the presentinvention are used to in T cell assays to determine the therapeuticpotential of compounds or biological agents to modulate PD-1-dependentregulation of T cell-dependent response and function. Exemplary T cellassays include, but are not limited to, ELISpot, intracellular cytokinestaining, major histocompatibility complex (MHC) restriction, viralsuppression assays, cytotoxicity assays, proliferation assays andregulatory T cell suppression assays.

In various embodiments, cells of non-human animals of the presentinvention are used in a cell transmigration assay to screen and developcandidate therapeutics that modulate human PD-1. Cell transmigrationinvolves the migration of cells across the endothelium andtransmigration assays permit the measurement of interactions with, andtransmigration of, the endothelium by leukocytes or tumor cells.

In various embodiments, cells of non-human animals of the presentinvention are used in tumor cell growth (or proliferation) assays todetermine the therapeutic potential of compounds or biological agents tomodulate PD-1-dependent regulation and/or apoptosis of tumor cells.

In various embodiments, cells of non-human animals of the presentinvention are used in cytokine production assays to determine thetherapeutic potential of compounds or biological agents to modulatePD-1-dependent regulation of cytokine release from T cells. In someembodiments, cells of non-human animals of the present invention areused for detection (and/or measurement) of intracellular cytokinerelease resulting from interaction of humanized PD-1 with a drugtargeting human PD-1 or a PD-1 ligand (e.g., PD-L1 or PD-L2).

In various embodiments, an autoimmune disease, disorder or condition isinduced in one or more non-human animals of the present invention toprovide an in vivo system for determining the therapeutic potential ofcompounds or biological agents to modulate PD-1-dependent regulation ofone or more functions of the autoimmune disease, disorder or condition.Exemplary autoimmune diseases, disorders or conditions that may beinduced in one or more non-human animals of the present inventioninclude diabetes, experimental autoimmune encephalomyelitis (e.g., amodel for multiple sclerosis), rheumatoid arthritis, and systemic lupuserythematosus.

Non-human animals of the present invention provide an in vivo system forthe analysis and testing of a drug or vaccine. In various embodiments, acandidate drug or vaccine may be delivered to one or more non-humananimals of the present invention, followed by monitoring of thenon-human animals to determine one or more of the immune response to thedrug or vaccine, the safety profile of the drug or vaccine, or theeffect on a disease or condition. In some embodiments, the vaccinetargets a virus such as, for example, human immunodeficiency virus orhepatitis virus (e.g. HCV). Exemplary methods used to determine thesafety profile include measurements of toxicity, optimal doseconcentration, efficacy of the drug or vaccine, and possible riskfactors. Such drugs or vaccines may be improved and/or developed in suchnon-human animals.

Non-human animals of the present invention provide an in vivo system forassessing the pharmacokinetic properties of a drug targeting human PD-1.In various embodiments, a drug targeting human PD-1 may be delivered oradministered to one or more non-human animals of the present invention,followed by monitoring of, or performing one or more assays on, thenon-human animals (or cells isolated therefrom) to determine the effectof the drug on the non-human animal. Pharmacokinetic properties include,but are not limited to, how an animal processes the drug into variousmetabolites (or detection of the presence or absence of one or more drugmetabolites, including, toxic metabolites), drug half-life, circulatinglevels of drug after administration (e.g., serum concentration of drug),anti-drug response (e.g., anti-drug antibodies), drug absorption anddistribution, route of administration, routes of excretion and/orclearance of the drug. In some embodiments, pharmacokinetic andpharmacodynamic properties of drugs (e.g., PD-1 modulators) aremonitored in or through the use of non-human animals of the presentinvention.

Non-human animals of the present invention provide an in vivo system forassessing the on-target toxicity of a drug targeting human PD-1. Invarious embodiments, a drug targeting human PD-1 may be delivered oradministered to one or more non-human animals of the present invention,followed by monitoring of or performing one or more assays on thenon-human animals (or cells isolated therefrom) to determine theon-target toxic effect of the drug on the non-human animal. Typically,drugs are intended to modulate one or more functions of their targets.To give but one example, a PD-1 modulator is intended to modulatePD-1-mediated functions (e.g., PD-1 signal transduction) throughinteracting in some way with the PD-1 molecule on the surface of one ormore cells. In some embodiments, such a modulator may have an adverseeffect that is an exaggeration of the desired pharmacologic action(s) ofthe modulator. Such effects are termed on-target effects. Exemplaryon-target effects include too high of a dose, chronicactivation/inactivation, and correct action in an incorrect tissue. Insome embodiments, on-target effects of a drug targeting PD-1 identifiedin or through the use of non-human animals of the present invention areused to determine a previously unknown function(s) of PD-1.

Non-human animals of the present invention provide an in vivo system forassessing the off-target toxicity of a drug targeting human PD-1. Invarious embodiments, a drug targeting human PD-1 may be delivered oradministered to one or more non-human animals of the present invention,followed by monitoring of or performing one or more assays on thenon-human animals (or cells isolated therefrom) to determine theoff-target toxic effect of the drug on the non-human animal. Off-targeteffects can occur when a drug interacts with an unintended target (e.g.,cross-reactivity to a common epitope). Such interactions can occur in anintended or unintended tissue. To give but one example, mirror imageisomers (enantiomers) of a drug can lead to off-target toxic effects.Further, a drug can inappropriately interact with and unintentionallyactivate different receptor subtypes. Exemplary off-target effectsinclude incorrect activation/inhibition of an incorrect targetregardless of the tissue in which the incorrect target is found. In someembodiments, off-target effects of a drug targeting human PD-1 aredetermined by comparing the effects of administering the drug tonon-human animals of the present invention to one or more referencenon-human animals.

In some embodiments, performing an assay includes determining the effecton the phenotype and/or genotype of the non-human animal to which thedrug is administered. In some embodiments, performing an assay includesdetermining lot-to-lot variability for a PD-1 modulator (e.g., anantagonist or an agonist). In some embodiments, performing an assayincludes determining the differences between the effects of a drugtargeting PD-1 administered to a non-human animal of the presentinvention and a reference non-human animal. In various embodiments,reference non-human animals may have a modification as described herein,a modification that is different as described herein (e.g., one that hasa disruption, deletion or otherwise non-functional Pdcd1 gene) or nomodification (i.e., a wild-type non-human animal).

Exemplary parameters that may be measured in non-human animals (or inand/or using cells isolated therefrom) for assessing the pharmacokineticproperties, on-target toxicity, and/or off-target toxicity of a drugtargeting human PD-1 include, but are not limited to, agglutination,autophagy, cell division, cell death, complement-mediated hemolysis, DNAintegrity, drug-specific antibody titer, drug metabolism, geneexpression arrays, metabolic activity, mitochondrial activity, oxidativestress, phagocytosis, protein biosynthesis, protein degradation, proteinsecretion, stress response, target tissue drug concentration, non-targettissue drug concentration, transcriptional activity and the like. Invarious embodiments, non-human animals of the present invention are usedto determine a pharmaceutically effective dose of a PD-1 modulator.

Non-human animals of the present invention provide an improved in vivosystem for the development and characterization of candidatetherapeutics for use in cancer. In various embodiments, non-humananimals of the present invention may be implanted with a tumor, followedby administration of one or more candidate therapeutics. In someembodiments, candidate therapeutics may include a multi-specificantibody (e.g., a bi-specific antibody) or an antibody cocktail; in someembodiments, candidate therapeutics include combination therapy such as,for example, administration of mono-specific antibodies dosedsequentially or simultaneously. The tumor may be allowed sufficient timeto be established in one or more locations within the non-human animal.Tumor cell proliferation, growth, survival, etc. may be measured bothbefore and after administration with the candidate therapeutic(s).Cytoxicity of candidate therapeutics may also be measured in thenon-human animal as desired.

Non-human animals of the present invention may be used to develop one ormore disease models to evaluate or assess candidate therapeutics and/ortherapeutic regimens (e.g., monotherapy, combination therapy, dose rangetesting, etc.) to effectively treat diseases, disorders or conditionsthat affect humans. Various disease conditions may be established innon-human animals of the present invention followed by administration ofone or more candidate molecules (e.g., drugs targeting PD-1) so thatefficacy of the one or more candidate molecules in a disease conditioncan determined. In some embodiments, disease models include autoimmune,inflammatory and/or neoplastic diseases, disorders or conditions.

To give but one example, non-human animals of the present inventionprovide an improved animal model for prophylactic and/or therapeutictreatment of a tumor or tumor cells. In various embodiments, non-humananimals of the present invention may be implanted with one or more tumorcells, followed by administration of one or more candidate therapeutics(e.g., antibodies). In some embodiments, administration of one or morecandidate therapeutics is performed subsequent to (e.g., minutes orhours but typically on the same day as) implantation of one or moretumor cells and one or more candidate therapeutics are evaluated innon-human animals of the present invention for efficacy in preventingestablishment of a solid tumor and/or growth of tumor cells in saidnon-human animals. In some embodiments, administration of one or morecandidate therapeutics is performed subsequent to (e.g., days after)implantation of one or more tumor cells and, in some certainembodiments, after a sufficient time such that one or more implantedtumor cells have reached a predetermined size (e.g., volume) innon-human animals of the present invention; and one or more candidatetherapeutics are evaluated for efficacy in treatment of one or moreestablished tumors. Non-human animals may be placed into differenttreatment groups according to dose so that an optimal dose or dose rangethat correlates to effective treatment of an established tumor can bedetermined.

Candidate molecules can be administered to non-human animal diseasemodels using any method of administration including parenteral andnon-parenteral routes of administration. Parenteral routes include,e.g., intravenous, intraarterial, intraportal, intramuscular,subcutaneous, intraperitoneal, intraspinal, intrathecal,intracerebroventricular, intracranial, intrapleural or other routes ofinjection. Non-parenteral routes include, e.g., oral, nasal,transdermal, pulmonary, rectal, buccal, vaginal, ocular. Administrationmay also be by continuous infusion, local administration, sustainedrelease from implants (gels, membranes or the like), and/or intravenousinjection. When a combination therapy is evaluated in non-human animalsof the present invention, candidate molecules can be administered viathe same administration route or via different administration routes.When a dosing regimen is evaluated in non-human animals of the presentinvention, candidate molecules may be administered at bimonthly,monthly, triweekly, biweekly, weekly, daily, at variable intervalsand/or in escalating concentrations to determine a dosing regimen thatdemonstrates a desired therapeutic or prophylactic effect in a non-humananimal in which one or more disease models has been established.

Non-human animals of the present invention provide an improved in vivosystem for the development and characterization of candidatetherapeutics for use in infectious diseases. In various embodiments,non-human animals of the present invention may be infected by injectionwith a virus (e.g., MHV, HIV, HCV, etc.) or pathogen (e.g., bacteria),followed by administration of one or more candidate therapeutics. Insome embodiments, candidate therapeutics may include a multi-specificantibody (e.g., a bi-specific antibody) or an antibody cocktail; in someembodiments, candidate therapeutics include combination therapy such as,for example, administration of mono-specific antibodies dosedsequentially or simultaneously; in some embodiments, candidatetherapeutics may include a vaccine. The virus or pathogen may be allowedsufficient time to be established in one or more locations or cellswithin the non-human animal so that one or more symptoms associated withinfection of the virus or pathogen develop in the non-human animal. Tcell proliferation and growth may be measured both before and afteradministration with the candidate therapeutic(s). Further, survival,serum and/or intracellular cytokine analysis, liver and/or spleenhistopathology may be measured in non-human animals infected with thevirus or pathogen. In some embodiments, non-human animals of the presentinvention are used to determine the extent of organ damage associatedwith viral infection. In some embodiments, non-human animals of thepresent invention are used to determine the cytokine expression profilein various organs of non-human animals infected with a particular virus.

Non-human animals of the present invention can be employed to assess theefficacy of a therapeutic drug targeting human cells. In variousembodiments, a non-human animal of the present invention is transplantedwith human cells, and a drug candidate targeting such human cells isadministered to such non-human animal. The therapeutic efficacy of thedrug is then determined by monitoring the human cells in the non-humananimal after the administration of the drug. Drugs that can be tested inthe non-human animals include both small molecule compounds, i.e.,compounds of molecular weights of less than 1500 kD, 1200 kD, 1000 kD,or 800 daltons, and large molecular compounds (such as proteins, e.g.,antibodies), which have intended therapeutic effects for the treatmentof human diseases and conditions by targeting (e.g., binding to and/oracting on) human cells.

In some embodiments, the drug is an anti-cancer drug, and the humancells are cancer cells, which can be cells of a primary cancer or cellsof cell lines established from a primary cancer. In these embodiments, anon-human animal of the present invention is transplanted with humancancer cells, and an anti-cancer drug is given to the non-human animal.The efficacy of the drug can be determined by assessing whether growthor metastasis of the human cancer cells in the non-human animal isinhibited as a result of the administration of the drug.

In specific embodiments, the anti-cancer drug is an antibody molecule,which binds an antigen on human cancer cells. In particular embodiments,the anti-cancer drug is a bi-specific antibody that binds to an antigenon human cancer cells, and to an antigen on other human cells, forexample, cells of the human immune system (or “human immune cells”) suchas B cells and T cells.

EXAMPLES

The following examples are provided so as to describe to those ofordinary skill in the art how to make and use methods and compositionsof the invention, and are not intended to limit the scope of what theinventors regard as their invention. Unless indicated otherwise,temperature is indicated in Celsius, and pressure is at or nearatmospheric.

Example 1 Humanization of an Endogenous Programmed Cell Death 1 (Pdcd1)Gene

This example illustrates exemplary methods of humanizing an endogenousPdcd1 gene encoding Programmed cell death protein 1 (PD-1) in anon-human mammal such as a rodent (e.g., a mouse). The methods describedin this example can be employed to humanize an endogenous Pdcd1 gene ofa non-human animal using any human sequence, or combination of humansequences (or sequence fragments) as desired. In this example, an ˜883bp human DNA fragment containing exon 2, intron 2, and the first 71 bpof exon 3 of a human PDCD1 gene that appears in GenBank accessionNM_005018.2 (SEQ ID NO:23) is employed for humanizing an endogenousPdcd1 gene of a mouse. A targeting vector for humanization of thegenetic material encoding an extracellular N-terminal IgV domain, of anendogenous Pdcd1 gene was constructed using VELOCIGENE® technology (see,e.g., U.S. Pat. No. 6,586,251 and Valenzuela et al., 2003, NatureBiotech. 21(6):652-659; herein incorporated by reference).

Briefly, mouse bacterial artificial chromosome (BAC) clone RP23-93N20(Invitrogen) was modified to delete the sequence containing exon 2,intron 2 and part of exon 3 of an endogenous Pdcd1 gene and insert exon2, intron 2 and part of exon 3 of a human PDCD1 gene using an ˜883 bphuman DNA fragment, which encodes amino acids 26-169 of a human PD-1polypeptide. Endogenous DNA containing exon 1, portion of exon 3 (i.e.,that encodes the transmembrane domain), 4 and 5 as well as the 5′ and 3′untranslated regions (UTRs) were retained. Sequence analysis of the ˜883bp human DNA fragment confirmed all human PDCD1 exons (i.e., exon 2 and71 bp of exon 3) and splicing signals. Sequence analysis revealed thatthe sequence matched the reference genome and PDCD1 transcriptNM_005018.2.

In more detail, first, a small bacterial homologous recombination donorwas constructed from a synthetic DNA fragment containing the following:[(HindIII)-(mouse upstream 78 bp)-(XhoI/NheI restriction enzymesites)-(human PDCD1 883 bp)-(mouse downstream 75 bp)-(HindIII)]. Thisfragment was synthesized by Genescript Inc. (Piscataway, N.J.) andcloned into an ampicillin-resistant plasmid vector. The XhoI-NheI siteswere employed to ligate a ˜4,996 bp self-deleting neomycin cassetteflanked by recombinase recognition sites(loxP-hUb1-em7-Neo-pA-mPrm1-Crei-loxP; see U.S. Pat. Nos. 8,697,851,8,518,392 and 8,354,389, which are herein incorporated by reference).Subsequent selection employed neomycin. The flanking HindIII sites wereused to linearize the targeting vector prior to homologous recombinationwith mouse BAC clone RP23-93N20. By design, the junction between theHuman PDCD1 883 bp fragment and the mouse downstream 75 bp preserved theopen reading frame in exon 3 (FIG. 2). The resulting targeting vectorcontained, from 5′ to 3′, a 5′ homology arm containing ˜61.7 kb of mousegenomic DNA from BAC clone RP23-93N20, a self-deleting neomycin cassetteflanked by loxP sites, an 883 bp human genomic DNA fragment (containingexon 2 through the first 71 bp of exon 3 of a human Pdcd1 gene) and ˜84kb of mouse genomic DNA from BAC clone RP23-93N20.

The modified RP23-93N20 BAC clone described above was used toelectroporate mouse embryonic stem (ES) cells to create modified EScells comprising an endogenous Pdcd1 gene that is humanized from exon 2through to part of exon 3 (i.e., deletion of 900 bp of the endogenousPdcd1 gene and insertion of 883 bp of human sequence). Positivelytargeted ES cells containing a humanized Pdcd1 gene were identified byan assay (Valenzuela et al., supra) that detected the presence of thehuman PDCD1 sequences (e.g., exon 2 and part of exon 3) and confirmedthe loss and/or retention of mouse Pdcd1 sequences (e.g., exon 2 andpart of exon 3, and/or exons 1, 4 and 5). Table 4 sets forth the primersand probes that were used to confirm humanization of an endogenous Pdcd1gene as described above (FIG. 3). The nucleotide sequence across theupstream insertion point included the following, which indicatesendogenous mouse sequence (contained within the parentheses below withan XhoI restriction site italicized) upstream of the 5′ end ofself-deleting neomycin cassette of the insertion point linkedcontiguously to a loxP site (bolded) and cassette sequence present atthe insertion point: (TCAAAGGACA GAATAGTAGC CTCCAGACCC TAGGTTCAGTTATGCTGAAG GAAGAGCCCT CTCGAG)ATAACTTCGT ATAATGTATG CTATACGAAG TTATATGCATGGCCTCCGCG CCGGGTTTTG GCGCCTCCCG CGGGCGCCCC CCTCCTCACG (SEQ ID NO:19).The nucleotide sequence across the downstream insertion point at the 3′end of the self-deleting neomycin cassette included the following, whichindicates cassette sequence (contained within the parentheses below withloxP sequence bolded and an NheI restriction site italicized) contiguouswith human Pdcd1 genomic sequence downstream of the insertion point:(CTGGAATAAC TTCGTATAAT GTATGCTATA CGAAGTTATG CTAGTAACTA TAACGGTCCTAAGGTAGCGA GCTAGC) AAGAGGCTCT GCAGTGGAGG CCAGTGCCCA TCCCCGGGTGGCAGAGGCCC CAGCAGAGAC TTCTCAATGA CATTCCAGCT GGGGTGGCCC TTCCAGAGCCCTTGCTGCCC GAGGGATGTG AGCAGGTGGC CGGGGAGGCT TTGTGGGGCC ACCCAGCCCC (SEQID NO:20). The nucleotide sequence across the downstream insertion pointat the 3′ end of the human PDCD1 genomic sequence included thefollowing, which indicates human PDCD1 sequence contiguous with mousePdcd1 genomic sequence (contained within the parentheses below):CCCTTCCAGA GAGAAGGGCA GAAGTGCCCA CAGCCCACCC CAGCCCCTCA CCCAGGCCAGCCGGCCAGTT CCAAACCCTG (GTCATTGGTA TCATGAGTGC CCTAGTGGGT ATCCCTGTATTGCTGCTGCT GGCCTGGGCC CTAGCTGTCT TCTGCTCAAC) (SEQ ID NO:21). Thenucleotide sequence across the upstream insertion point after deletionof the neomycin cassette (77 bp remaining) included the following, whichindicates mouse and human genomic sequence juxtaposed with remainingcassette sequence loxP sequence (contained within the parentheses belowwith XhoI and NheI restriction sites italicized and loxP sequence inbold): TCAAAGGACA GAATAGTAGC CTCCAGACCC TAGGTTCAGT TATGCTGAAG GAAGAGCCCT(CTCGAG ATAACTTCGT ATAATGTATG CTATACGAAG TTATGCTAGT AACTATAACGGTCCTAAGGT AGCGA GCTAGC) AAGAG GCTCTGCAGT GGAGGCCAGT GCCCATCCCCGGGTGGCAGA GGCCCCAGCA GAGACTTCTC AATGACATTC CAGCTGGGGT GGCCCTTCCA (SEQID NO:22).

Positive ES cell clones were then used to implant female mice using theVELOCIMOUSE® method (see, e.g., U.S. Pat. No. 7,294,754 and Poueymirouet al., 2007, Nature Biotech. 25(1):91-99) to generate a litter of pupscontaining an insertion of human PDCD1 exon 2 and part of human PDCD1exon 3 into an endogenous Pdcd1 gene of a mouse. Mice bearing thehumanization of exon 2 and 3 in part (i.e., the 883 bp human DNAfragment) of an endogenous Pdcd1 gene were again confirmed andidentified by genotyping of DNA isolated from tail snips using amodification of allele assay (Valenzuela et al., supra) that detectedthe presence of the human PDCD1 gene sequences. Pups are genotyped andcohorts of animals heterozygous for the humanized Pdcd1 gene constructare selected for characterization.

TABLE 4 Sequence Name Primer (5′-3′) 7106 hTU Forward CCCAGCAGAGACTT(SEQ ID NO: 7) CTCAATGAC Probe TGGCCCTTCCAGAG (SEQ ID NO: 8) CCCTTGReverse CGGCCACCTGCTCA (SEQ ID NO: 9) CATC 7106 hTD ForwardGGCATCTCTGTCCT (SEQ ID NO: 10) CTAGCTC Probe AAGCACCCCAGCCC(SEQ ID NO: 11) CTCTAGTCTG Reverse GGGCTGTGGGCACT (SEQ ID NO: 12) TCTG7106 TU Forward CCTTCCTCACAGCT (SEQ ID NO: 13) CTTTGTTC ProbeTCTGCATTTCAGAG (SEQ ID NO: 14) GTCCCCAATGG Reverse GAGCCAGGCTGGGT(SEQ ID NO: 15) AGAAG 7106 TD Forward CGGTGTCCTAGAAC (SEQ ID NO: 16)TCTATTCTTTG Probe TCCTGGAGACCTCA (SEQ ID NO: 17) ACAAGATATCCCA ReverseTGAAACCGGCCTTC (SEQ ID NO: 18) TGGTT

Example 2 Expression of Humanized PD-1 on Activated T Cells

This Example demonstrates that non-human animals (e.g., rodents)modified to contain a humanized Pdcd1 gene according to Example 1express a humanized PD-1 protein on the surface of activatedlymphocytes. In this Example, activated T cells from mice heterozygousfor humanization of an endogenous Pdcd1 gene as described in Example 1were stained with anti-PD-1 antibodies to determine the expression ofPD-1 in stimulated T cells isolated from wild-type and humanized mice.

Briefly, spleens were harvested and processed from a wild-type mouse anda mouse heterozygous for humanization of an endogenous Pdcd1 gene asdescribed in Example 1 into single cell suspensions by mechanicaldissociation. Cells were washed in media (RPMI supplemented with 10%FBS) and re-suspended at 1×10⁶/mL and 200 μL (200,000 cells) were platedin 96-well plates. Cells in selected wells were stimulated with anti-CD3and anti-CD28 antibodies (both at 1 μg/mL) for 72 hours. Cells werestained for FACS according to manufacturer's specifications withantibodies recognizing CD4, CD8, CD19 and human (clone MIH4, BDBiosciences) or mouse (clone J43, eBioscience) PD1. Stained cells wereran on LSRII flow cytometer and data was analyzed using Flowjo software.CD8⁺ T cells were gated (CD19⁻CD8⁺) for expression of human and mousePD1. Exemplary results are shown in FIG. 4.

As shown in FIG. 4, mice bearing a humanized Pdcd1 gene as described inExample 1 express a PD-1 polypeptide that comprises a human portion andan endogenous mouse portion. The human portion is detectably expressedvia recognition by an antibody that recognizes a fully human PD-1polypeptide.

Example 3 In Vivo Efficacy of PD-1 Modulators

This Example demonstrates that non-human animals (e.g., rodents)modified to contain a humanized Pdcd1 gene according to Example 1 can beused in an in vivo assay to screen PD-1 modulators (e.g., anti-PD-1antibodies) and determine various characterisitics such as, for example,inhibition of tumor growth and/or killing of tumor cells. In thisExample, several anti-PD-1 antibodies are screened in mice homozygousfor humanization of an endogenous Pdcd1 gene as described in Example 1to determine the optimal antibody dose that inhibits tumor growth andthe extent to which anti-PD-1 antibodies mediate killing of tumor cells.

Briefly, mice were divided evenly according to body weight into fivetreatment or control groups for Study 1 (n=5/group), eight treatment orcontrol groups for Study 2 (n=5/group), and five treatment or controlgroups for Study 3 (n=7/group). At day zero, mice were anesthetized byisoflurane inhalation and then subcutaneously injected with MC38.ovacells in suspension of 100 μL of DMEM into the right flank (Study 1:5×10⁵; Study 2/3: 1×10⁶). MC38.ova (mouse colon adenocarcinoma) cellswere engineered to express chicken ovalbumin in order to increase tumorimmunogenicity. For Study 1, treatment groups were intraperitoneallyinjected with 200 μg of either one of three anti-PD-1 antibodies, or anisotype control antibody with irrelevant specificity on days 3, 7, 10,14, and 17 of the experiment, while one group of mice was leftuntreated. For Study 2, treatment groups were intraperitoneally injectedwith either one of three anti-PD-1 antibodies at 10 mg/kg or 5 mg/kgper/dose, one anti-PD-1 antibody (Ab B, IgG4) at 10 mg/kg per dose, oran isotype control antibody with irrelevant specificity at 10 mg/kg ondays 3, 7, 10, 14, and 17 of the experiment. For Study 3, treatmentgroups were intraperitoneally injected with either one of two anti-PD-1antibodies at 5 mg/kg or 2.5 mg/kg per/dose, or a control antibody notspecific to PD-1 (control) at 5 mg/kg on days 3, 7, 10, 14, and 17 ofthe experiment. Table 5 sets forth experimental dosing and treatmentprotocol for groups of mice.

For each of the studies, average tumor volumes determined by calipermeasurements and percent survival at Day 14 or 17 and Day 23 or 24 ofeach experiment for each treatment group were recorded. The number oftumor-free mice were also assessed at the end of the study (Day 42 forStudy 1 and Day 31 for Study 2 and Study 3). Mean tumor volume(mm³)(±SD), percent survival, and number of tumor-free mice werecalculated for each study (Tables 7-9). Exemplary tumor growth curvesare provided in FIG. 5.

As shown in Table 6 for Study 1, mice treated with Ab A did not developany detectable tumors during the course of the study. Mice treated withAb C exhibited a sustained reduced tumor volume as compared to controlsat days 17 and 24 of the study; and 3 out of 5 mice were tumor free bythe end of the experiment. In contrast, treatment with Ab B did notdemonstrate significant efficacy in reducing tumor volume in this studyas compared to controls. By day 23 of the study, 1 out of 5 mice died inthe group that received Ab B, and 2 out of 5 mice died in the isotypecontrol treatment group. In non-treatment and isotype control groups,some mice exhibited spontaneous regression of tumors (1 out of 5 miceand 2 out of 5 mice, respectively).

As shown in Table 7 for Study 2, mice treated with Ab A at 10 mg/kg didnot develop detectable tumors during the course of the study. Groups ofmice treated with 10 mg/kg of either Ab C or Ab D exhibitedsubstantially reduced tumor volume as compared to controls at days 17and 24 of the study. Four out of 5 mice in each group treated with 10mg/kg of either Ab C or Ab D were tumor free at Day 31, whereas in theisotype control treatment group only 1 out of 5 animals was tumor freeas a result of spontaneous tumor regression. Ab B tested at 10 mg/kgdemonstrated substantially reduced tumor volume as compared to controlsat days 17 and 24 of the study, but this antibody was the leastefficacious anti-PD1 antibody with only 2 out of 5 mice surviving at theend of the experiment.

A dose-dependent response in tumor suppression at the tested doses (5mg/kg and 10 mg/kg) was observed in groups treated with Ab A, Ab C, andAb D. Ab A or Ab C therapy at 5 mg/kg was less efficacious, with 4 outof 5 tumor-free mice at the end of experiment on day 31, whereas 5 outof 5 mice remained tumor-free in 10 mg/kg dose group of Ab A. Dunett'stest in 2 way ANOVA multiple comparisons revealed that the differencesin tumor growth between the group treated with isotype control antibodyat 10 mg/kg as reference and the groups treated at 10 mg/kg with Ab A,Ab C or Ab D were statistically significant with p value <0.005. Thedifferences in tumor growth between the group treated with isotypecontrol antibody at 10 mg/kg as reference and the groups treated at 5mg/kg with Ab A, Ab C or Ab D were also statistically significant with ap value <0.05.

As shown in Table 8 for Study 3, 6 out or 7 mice treated with Ab A or AbC at 5 mg/kg were tumor free at the end of the experiment, whereas therewere no tumor free animals in the isotype control group. Onetumor-bearing mouse in the IgG4 control group died on post-implantationday 17. Only 4 out of 7 mice treated with Ab C at 2.5 mg/kg remainedtumor free at the end of the experiment. The difference in tumor volumesat day 21 between anti-PD-1 antibodies tested and an isotype controlgroup was statistically significant as determined by one-way ANOVA withDunnett's multiple comparison post-test with p<0.01. All four anti-PD-1antibodies tested were equally more efficacious at the 5 mg/kg dose thanat the 2.5 mg/kg dose.

As shown in FIG. 5, anti-PD-1 antibodies significantly inhibited tumorgrowth in a prophylactic MC38.ova tumor growth model in PD-1 humanizedmice made according to Example 1. Anti-PD-1 Ab therapy at 10 mg/kgpromoted tumor regression in all mice (5 out of 5) throughout the courseof the experiment, whereas only one out of five animals remainedtumor-free in the control group resulting from spontaneous tumorregression. Anti-PD-1 therapy at 5 mg/kg was slightly less efficacious,with four out of five tumor-free mice at the end of the experiment.One-way ANOVA with Dunnett's multiple comparison post-test revealed asignificant difference in tumor volumes between anti-PD-1 and controlantibody treatments with a p value <0.05 (5 mg/kg) and p value <0.01 (10mg/kg).

In a similar experiment, intact functional PD-1 signaling in PD-1humanized mice made according to Example 1 was investigated by measuringCD8⁺ T cells and CD3⁺ T cells responses and IFNγ production in spleensof tumor-bearing mice treated with anti-PD-1 antibody.

Briefly, spleen cells were obtained from PD-1 humanized mice (75%C57BL/6/25% 129) treated with anti-PD-1 or control antibody at the endof the experiments on Day 21 (described above). Total RNA was isolated,and real-time PCR was performed on reverse transcribed cDNA usingoligonucleotides and taqman probe mix specific for mouse CD8b (forwardprimer: GCTCTGGCTG GTCTTCAGTA TG, SEQ ID NO:24; reverse primer:TTGCCGTATG GTTGGTTTGA AC, SEQ ID NO:25; probe: AGCAGCTCTG CCCTCAT, SEQID NO:26), mouse CD3ζ (Mm00446171_m1, Applied Biosystems), mouse IFN-γ(Mm01168134_m1, Applied Biosystems), human PD-1 (forward primer:ACTTCCACAT GAGCGTGG, SEQ ID NO:27; reverse primer: GGGCTGTGGG CACTTCTG,SEQ ID NO:28; probe: GCAGATCAAA GAGAGCCTGC, SEQ ID NO:29) and mouse PD-1(Mm01285676_m1, Applied Biosystems). Samples were normalized relative toexpression of mouse cyclophilin B. Exemplary results are provided inFIG. 6.

As shown in FIG. 6, administration of anti-hPD-1 antibody inducedincreased production of CD8⁺ and CD3⁺ T cells in spleens of humanizedmice (made according to Example 1) bearing MC38.ova tumors. Further,activity of anti-hPD-1 antibody in tumor bearing PD-1 humanized mice wasdependent on IFNγ, which confirmed proper signaling through humanizedPD-1 on the cell surface. Overall, an increase in T cells and IFNγ ascompared to control-treated mice was observed for both treatment groups.

Human PD-1 mRNA expression was measured with human specific probesdesigned for the extracellular portion of the PD-1 protein and confirmedproper expression of humanized PD-1 protein on the cell surface.Additionally, measurement of mouse PD-1 mRNA expression with primersdesigned to detect the extracellular portion of mouse PD-1 failed toproduce a product.

Taken together, this Example demonstrates that non-human animals of thepresent invention can be used to assess the in vivo efficacy of drugs(e.g., an antibody) targeting PD-1, and such animals are useful indiscriminating the therapeutic effect of anti-PD-1 antibodies. Moreover,non-human animals described herein can be used to assess the extent towhich drugs targeting PD-1 can inhibit tumor growth and/or mediatekilling of tumor cells. Non-human animals (e.g., mice) of the presentinvention demonstrate functional PD-1-signaling and properPD-1-dependent immune responses via humanized PD-1 as evidenced byexpansion of T cells and cytokine expression (e.g., IFN-γ).

TABLE 5 Study # Antibody Dosage 1 Isotype Control 200 μg No treatmentN/A Ab A 200 μg Ab B 200 μg Ab C 200 μg 2 Isotype Control 10 mg/kg Ab A10 mg/kg Ab A 5 mg/kg Ab B 10 mg/kg Ab C 10 mg/kg Ab C 5 mg/kg Ab D 10mg/kg Ab D 5 mg/kg 3 Isotype Control 5 mg/kg Ab A 5 mg/kg Ab A 2.5 mg/kgAb C 5 mg/kg Ab C 2.5 mg/kg

TABLE 6 Study 1 Mean tumor volume Tumor free (mm³, ±SD) Survival (%)mice Treatment Day 17 Day 23 Day 17 Day 23 Day 42 group 200 μg/ 200 μg/200 μg/ 200 μg/ 200 μg/ (n = 5) mouse mouse mouse mouse mouse Notreatment 189 (±110) 554 (±317) 100% 100% 1/5 Isotype Control 86 (±114)515 (±859) 100% 60% 2/5 Ab A 0 (0) 0 (0) 100% 100% 5/5 Ab B 89 (±176)445 (±889) 100% 80% 3/5 Ab C 14 (±19) 205 (±312) 100% 100% 3/5

TABLE 7 Study 2 Mean tumor volume Tumor free Treatment (mm³; ±SD)Survival (%) Mice group Day 17 Day 24 Day 17 Day 24 Day 31 (n = 5) 5mg/kg 10 mg/kg 5 mg/kg 10 mg/kg 5 mg/kg 10 mg/kg 5 mg/kg 10 mg/kg 5mg/kg 10 mg/kg Isotype N/A 449 (±434) N/A 824 (±858) N/A 100% N/A 60%N/A 1/5 Control Ab A 17 (±38)  0 (0) 104 (±233)  0 (0) 100 100 100  1004/5 5/5 Ab B N/A 124 (±209) N/A 359 (±657) N/A 100 N/A  80 N/A 2/5 Ab C91 (±204)  12 (±28) 228 (±509)  96 (±215) 100 100 80 100 4/5 4/5 Ab D 94(±160)  10 (±21) 328 (±559)  67 (±150) 100 100 80 100 3/5 4/5

TABLE 8 Study 3 Mean tumor volume Tumor free (mm³; ±SD) Survival (%)mice Treatment Days 14 Day 21 Day 14 Day 21 Day 31 group (n = 7) 2.5mg/kg 5 mg/kg 2.5 mg/kg 5 mg/kg 2.5 mg/kg 5 mg/kg 2.5 mg/kg 5 mg/kg 2.5mg/kg 5 mg/kg Isotype N/A 94 (±44) N/A 405 (±326) N/A 100 N/A 86 N/A 0/7Control Ab A  0 (0)  0 (0) 19 (±51)  13 (±35) 100 100 100 100 6/7 6/7 AbC 41 (±68)  7 (±20) 87 (±123)  16 (±42) 100 100 100 100 4/7 6/7

Example 4 Rodent Model of Anti-PD-1 Tumor Therapy

This Example demonstrates that non-human animals (e.g., rodents)modified to contain a humanized Pdcd1 gene according to Example 1 can beused in a tumor model to determine optimal therapeutic dose(s) of PD-1modulators (e.g., anti-PD-1 antibodies). In this Example, an anti-PD-1antibody is administered to mice homozygous for humanization of anendogenous Pdcd1 gene as described in Example 1 to determine the optimaltherapeutic dose for treatment of established tumors.

Briefly, mice containing a humanized Pdcd1 gene (as described inExample 1) were subcutaneously implanted with 1×10⁶ MC38.Ova cells(described above) and subsequently randomized into six treatment groups(n=8-9 per group) once tumor volumes reached 80-120 mm³ (day 0). Micewere intraperitoneally administered anti-hPD-1 antibody in an escalatingdose range of 0.3-25 mg/kg (i.e., 0.3, 1, 3, 10 or 25 mg/kg) or anisotype control antibody at 25 mg/kg. Antibodies were dosed on days 0,3, 7, 10 and 13. Tumor volumes were monitored by calipered measurementstwice per week for the duration of the experiment (60 days). Exemplarytumor growth curves are provided in FIG. 7.

As shown in FIG. 7, none of the mice administered the control antibodywere tumor free at the end of the experiment. In contrast, a dose rangeof 3-25 mg/kg anti-hPD-1 antibody resulted in about 44-55% tumor freemice among the different treatment groups. Taken together, this Exampledemonstrates that non-human animals of the present invention can be usedas a rodent tumor model to determine the optimal dose and/or dose rangeof drugs (e.g., an antibody) targeting PD-1 to effectively treatestablished tumors.

EQUIVALENTS

Having thus described several aspects of at least one embodiment of thisinvention, it is to be appreciated by those skilled in the art thatvarious alterations, modifications, and improvements will readily occurto those skilled in the art. Such alterations, modifications, andimprovements are intended to be part of this disclosure, and areintended to be within the spirit and scope of the invention.Accordingly, the foregoing description and drawing are by way of exampleonly and the invention is described in detail by the claims that follow.

Use of ordinal terms such as “first,” “second,” “third,” etc., in theclaims to modify a claim element does not by itself connote anypriority, precedence, or order of one claim element over another or thetemporal order in which acts of a method are performed, but are usedmerely as labels to distinguish one claim element having a certain namefrom another element having a same name (but for use of the ordinalterm) to distinguish the claim elements.

The articles “a” and “an” as used herein in the specification and in theclaims, unless clearly indicated to the contrary, should be understoodto include the plural referents. Claims or descriptions that include“or” between one or more members of a group are considered satisfied ifone, more than one, or all of the group members are present in, employedin, or otherwise relevant to a given product or process unless indicatedto the contrary or otherwise evident from the context. The inventionincludes embodiments in which exactly one member of the group is presentin, employed in, or otherwise relevant to a given product or process.The invention also includes embodiments in which more than one, or theentire group members are present in, employed in, or otherwise relevantto a given product or process. Furthermore, it is to be understood thatthe invention encompasses all variations, combinations, and permutationsin which one or more limitations, elements, clauses, descriptive terms,etc., from one or more of the listed claims is introduced into anotherclaim dependent on the same base claim (or, as relevant, any otherclaim) unless otherwise indicated or unless it would be evident to oneof ordinary skill in the art that a contradiction or inconsistency wouldarise. Where elements are presented as lists, (e.g., in Markush group orsimilar format) it is to be understood that each subgroup of theelements is also disclosed, and any element(s) can be removed from thegroup. It should be understood that, in general, where the invention, oraspects of the invention, is/are referred to as comprising particularelements, features, etc., certain embodiments of the invention oraspects of the invention consist, or consist essentially of, suchelements, features, etc. For purposes of simplicity those embodimentshave not in every case been specifically set forth in so many wordsherein. It should also be understood that any embodiment or aspect ofthe invention can be explicitly excluded from the claims, regardless ofwhether the specific exclusion is recited in the specification.

Those skilled in the art will appreciate typical standards of deviationor error attributable to values obtained in assays or other processesdescribed herein. The publications, websites and other referencematerials referenced herein to describe the background of the inventionand to provide additional detail regarding its practice are herebyincorporated by reference.

We claim:
 1. A mouse whose genome comprises a humanized Programmed celldeath 1 (Pdcd1) gene at an endogenous Pdcd1 locus, wherein the humanizedPdcd1 gene comprises exon 2, and comprises exon 3 in whole or in part,of a human Pdcd1 gene, wherein the humanized Pdcd1 gene is operablylinked to a Pdcd1 promoter and expresses a humanized Programmed celldeath 1 (PD-1) polypeptide on the surface of activated T cells in themouse, and wherein the humanized PD-1 polypeptide comprises theN-terminal immunoglobulin V domain of a human PD-1 polypeptide and theintracellular domain of an endogenous mouse PD-1 polypeptide.
 2. Themouse of claim 1, wherein the humanized PD-1 polypeptide is translatedin a cell of the mouse with a mouse signal peptide.
 3. The mouse ofclaim 1, wherein the humanized PD-1 polypeptide comprises thetransmembrane sequence of the endogenous PD-1 polypeptide.
 4. The mouseof claim 1, wherein the humanized PD-1 polypeptide comprises amino acids26-169 of a human PD-1 polypeptide.
 5. The mouse of claim 1, wherein thehumanized Pdcd1 gene comprises endogenous Pdcd1 exons 1, 4 and
 5. 6. Themouse of claim 5, wherein the humanized Pdcd1 gene further comprises anendogenous Pdcd1 exon 3 in part.
 7. The mouse of claim 1 wherein thePdcd1 promoter is a rodent Pdcd1 promoter.
 8. The mouse of claim 1,wherein the Pdcd1 promoter is an endogenous mouse Pdcd1 promoter.
 9. Themouse of claim 1, wherein the humanized Pdcd1 gene comprises thenucleotide sequence as set forth in SEQ ID NO:
 23. 10. The mouse ofclaim 1, wherein the mouse is homozygous for the humanized Pdcd1 gene.11. The mouse of claim 1, wherein the mouse does not detectably expressa full length endogenous mouse PD-1 polypeptide.
 12. A humanized PD-1polypeptide produced by the mouse of claim
 11. 13. The mouse of claim 1,wherein the humanized Pdcd1 gene comprises the part of exon 3 of thehuman Pdcd1 gene that encodes amino acids of the extracellular domain ofthe human PD-1 polypeptide.
 14. The mouse of claim 13, wherein thehumanized Pdcd1 gene comprises endogenous mouse Pdcd1 exon 1, exon 3 inpart, exon 4 and exon
 5. 15. The mouse of claim 13, wherein thehumanized PD-1 polypeptide comprises the transmembrane and intracellulardomains of the endogenous mouse PD-1 polypeptide.